Fibronectin based scaffold domain proteins that bind IL-23

ABSTRACT

The present invention relates to fibronectin based scaffold domain protein that bind interleukin 23 (IL-23), specifically the p19 subunit of IL-23. The invention also relates to the use of the innovative proteins in therapeutic applications to treat autoimmune diseases. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative proteins.

FIELD OF THE INVENTION

The present invention relates to fibronectin based scaffold domainprotein that bind interleukin 23 (IL-23), specifically the p19 subunitof IL-23. The invention also relates to the use of the innovativeproteins in therapeutic applications to treat autoimmune diseases. Theinvention further relates to cells comprising such proteins,polynucleotide encoding such proteins or fragments thereof, and tovectors comprising the polynucleotides encoding the innovative proteins.

INTRODUCTION

IL-23 is a member of the IL-12 heterodimeric cytokine family. Itcontains the p40 subunit, which is common to IL-12, and a unique p19subunit. IL-23 sends signals through a heterodimeric receptor complexconsisting of IL-12Rβ1 and IL-23R (Aggarwal, S et al., “Interleukin-23promotes a distinct CD4 T cell activation state characterized by theproduction of interleukin-17”, J. Biol. Chem., 278:1910-1914 (2003)).IL-23 is a potential target for the treatment of chronic inflammatorydisorders such as multiple sclerosis, rheumatoid arthritis, psoriasisand Crohn's disease.

Fibronectin based scaffolds are a family of proteins capable of evolvingto bind any compound of interest. These proteins, which generally makeuse of a scaffold derived from a fibronectin type III (Fn3) or Fn3-1 ikedomain, function in a manner characteristic of natural or engineeredantibodies (that is, polyclonal, monoclonal, or single-chain antibodies)and, in addition, possess structural advantages. Specifically, thestructure of these antibody mimics has been designed for optimalfolding, stability, and solubility, even under conditions that normallylead to the loss of structure and function in antibodies. An example offibronectin-based scaffold proteins are Adnectins (Adnexus, aBristol-Myers Squibb R&D Company).

Fibronectin type III (Fn3) domains comprise, in order from N-terminus toC-terminus, a beta or beta-like strand, A; a loop, AB; a beta orbeta-like strand, B; a loop, BC; a beta or beta-like strand C; a loopCD; a beta or beta-like strand D; a loop DE; a beta or beta-like strand,E; a loop, EF; a beta or beta-like strand F; a loop FG; and a beta orbeta-like strand G. Any or all of loops AB, BC, CD, DE, EF and FG mayparticipate in target binding. The BC, DE, and FG loops are bothstructurally and functionally analogous to the complementaritydetermining regions (CDRs) from immunoglobulins. U.S. Pat. No. 7,115,396describes Fn3 domain proteins wherein alterations to the BC, DE, and FGloops result in high affinity TNFα binders. U.S. Publication No.2007/0148126 describes Fn3 domain proteins wherein alterations to theBC, DE, and FG loops result in high affinity VEGFR2 binders.

It would be advantageous to obtain improved fibronectin domain scaffoldproteins for therapeutic treatment of autoimmune disorders. A subset ofeffector T cells that produce interleukin 17 (IL-17; “Th17 cells”) arehighly proinflammatory and induce severe autoimmunity. Th17 cellsexpress a distinct subset of cytokines and chemokines compared to Th1and Th2 cells, including IL-6, tumor necrosis factor (TNF), IL-22,IL-17A and IL-17F as well as the chemokine receptor CCR6. IL-23 promotesthe production of IL-17 by activated T cells (Aggarwal, S et al.,“Interleukin-23 promotes a distinct CD4 T cell activation statecharacterized by the production of interleukin-17”, J. Biol. Chem.,278:1910-1914 (2003)) and is a key cytokine to induce expansion ofIL-17-producing CD4+ T cells. Exposure to IL-23 seems to be the keyfeature that determines the pathogenicity of Th17 cells.

SUMMARY OF THE INVENTION

The application provides Adnectins™ against human IL-23-specific p19subunit. One aspect of the invention provides for polypeptidescomprising Fn3 domain in which one or more of the solvent accessibleloops has been randomized or mutated. In some embodiments, the Fn3domain is a Fn3 domain derived from the wild-type tenth module of thehuman fibronectin type III domain (¹⁰Fn3). In some embodiments, the¹⁰Fn3 polypeptide of the invention is at least 40%, 50%, 60%, 65%, 70%,75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3 domain.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop.

In some embodiments, the polypeptides of the invention comprises a tenthfibronectin type III (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain comprisesa loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loopFG; and has at least one loop selected from loop BC, DE, and FG with analtered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain.

In some embodiments, the polypeptide of the invention comprises a Fn3domain that comprises an amino acid sequence at least 80, 85, 90, 95,98, 99 or 100% identical to the non-loop regions.

In some embodiments, the BC loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2-6.

In some embodiments, the DE loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 7-48.

In some embodiments, the FG loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 49-59.

In some embodiments, the ¹⁰Fn3 domain may begin and/or end with aminoacid substitutions, insertions or deletions.

In some embodiments, the protein of the invention comprises one loopsequence from the BC loop sequences shown in SEQ ID NOs: 2-6, one DEloop sequence shown in SEQ ID NOs: 7-48 and one FG loop sequence shownin SEQ ID NOs: 49-59.

In some embodiments, the protein of the invention comprises a BC, DE andFG loop amino acid sequence at least 70, 75, 80, 85, 90, 95, 98, 99 or100% identical to of any one of SEQ ID NOS:2-59.

In some embodiments, the anti-IL-23 Adnectin comprises the amino acidsequence of any one of SEQ ID NOS:60-100.

In some embodiments, the anti-IL-23 Adnectin comprises the Fn3 domainamino acid sequence from position 3-96 of any one of SEQ ID NOS:60-100.

In some embodiments, the anti-IL-23 Adnectin comprises the amino acidsequence at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% identical to ofany one of SEQ ID NOS:60-100.

In one aspect, the anti-IL-23 Adnectin further comprising apharmacokinetic (PK) moiety. In some embodiments, the PK moietycomprises polyethylene glycol (PEG).

In one aspect, the application provides an anti-IL-23 Adnectin useful inthe treatment of autoimmune diseases.

In one aspect, the present invention provides a fusion polypeptidecomprising a fibronectin type III tenth (¹⁰Fn3) domain and anti-IL-23Adnectin, wherein the ¹⁰Fn3 domain binds to HSA with a Kd of 1 uM orless. In certain embodiments, the ¹⁰Fn3 domain comprises an amino acidsequence at least 70% identical to SEQ ID NO: 103. In one embodiment,the ¹⁰Fn3 domain comprises a BC loop having the amino acid sequence setforth in SEQ ID NO: 104, a DE loop having the amino acid sequence setforth in SEQ ID NO: 105, and an FG loop having the amino acid sequenceset forth in SEQ ID NO:106. In another embodiment, the ¹⁰Fn3 domaincomprises one or more of a BC loop having the amino acid sequence setforth in SEQ ID NO: 104, a DE loop having the amino acid sequence setforth in SEQ ID NO: 105, and an FG loop having the amino acid sequenceset forth in SEQ ID NO: 106.

In one embodiment, the ¹⁰Fn3 domain of the fusion polypeptide also bindsto one or more of rhesus serum albumin (RhSA), cynomolgus monkey serumalbumin (CySA), or murine serum albumin (MuSA). In other embodiments,the ¹⁰Fn3 domain does not cross-react with one or more of RhSA, CySA orMuSA.

In certain embodiments, the ¹⁰Fn3 domain of the fusion polypeptide bindsto HSA with a Kd of 1 uM or less. In some embodiments, the ¹⁰Fn3 domainbinds to HSA with a Kd of 500 nM or less. In other embodiments, the¹⁰Fn3 domain binds to HSA with a Kd of at least 200 nM, 100 nM, 50 nM,20 nM, 10 nM, or 5 nM.

In other embodiments, the ¹⁰Fn3 domain of the fusion polypeptide bindsto domain I or II of HSA. In one embodiment, the ¹⁰Fn3 domain binds toboth domains I and II of HSA. In some embodiments, the ¹⁰Fn3 domainbinds to HSA at a pH range of 5.5 to 7.4. In other embodiments, the¹⁰Fn3 domain binds to HSA with a Kd of 200 nM or less at pH 5.5. Inanother embodiment, the ¹⁰Fn3 domain binds to HSA with a Kd of at least500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 5 nM at a pH range of5.5 to 7.4. In one embodiment, the ¹⁰Fn3 domain binds to HSA with a Kdof at least 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 5 nM at pH5.5.

In some embodiments, the serum half-life of the fusion polypeptide inthe presence of serum albumin is at least 5-fold greater than the serumhalf-life of the polypeptide in the absence of serum albumin. In certainembodiments, the serum half-life of the fusion polypeptide in thepresence of serum albumin is at least 2-fold, 5-fold, 7-fold, 10-fold,12-fold, 15-fold, 20-fold, 22-fold, 25-fold, 27-fold, or 30-fold greaterthan the serum half-life of the polypeptide in the absence of serumalbumin. In some embodiments, the serum albumin is any one of HSA, RhSA,CySA, or MuSA.

In certain embodiments, the serum half-life of the fusion polypeptide inthe presence of serum albumin is at least 20 hours. In certainembodiments, the serum half-life of the fusion polypeptide in thepresence of serum albumin is at least 10 hours, 12 hours, 15 hours, 20hours, 25 hours, 30 hours, 40 hours, 50 hours, 75 hours, 90 hours, 100hours, 110 hours, 120 hours, 130 hours, 150 hours, 170 hours, or 200hours. In some embodiments, the half-life of the fusion polypeptide isobserved in a primate (e.g., human or monkey) or a murine.

In any of the foregoing aspects and embodiments, the ¹⁰Fn3 domaincomprises a sequence selected from SEQ ID NO: 107, 111, 115, 119, and123-143.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the full length DNA sequence alignment of the anti-IL23Adnectin of the invention.

FIG. 2 shows pBMS2008/ATI001044 protein expression vector as describedin Example 2.

FIG. 3 shows the full length amino acid sequence alignment of theanti-IL23 adnectin of the invention.

FIG. 4 shows a representative IC₅₀ curves from PBMC pSTAT3 inhibition byanti-IL-23 adnectin as described in Example 4.

FIG. 5 shows a representative IC₅₀ curves for inhibition ofIL-23-dependent IL-17A by anti-IL-23 adnectins and anti-p40 monoclonalantibody (MAB1510) as described in Example 4.

FIG. 6 shows ATI001045 inhibition of IL-23-induced IL-17 production byPBMCs of donor 228 (one of 4 donors tested as described in Example 4).

FIG. 7 shows representative selectivity data for the anti-IL-23adnectins. Buffer subtracted sensorgrams illustrating the associationand dissociation phases of 10 nM IL-23 and 1 uM IL-12 binding tocaptured ATI001016 as described in Example 4 are shown.

FIG. 8 shows that anti-IL-23 adnectins do not inhibit IL-12 inducedIFN-γ production in NK-92 cells as described in Example 4.

FIG. 9A shows that ATI001045 Inhibits Serum IL-17 Levels in MousePharmacodynamic Model as described in Example 4.

FIG. 9B shows a comparison of inhibitory activities of anti-IL-23adnectins in a mouse pharmacodynamic model as described in Example 4.

FIG. 10A shows ATI000934 dose response in human IL-23 induced acanthosisas described in Example 4.

FIG. 10B shows ATI001045 Dose Response in Human IL-23-Induced acanthosisas described in Example 4.

FIG. 11 shows in vivo HSA half-life in mice. HSA was injected into miceat 20 mg/kg (FIG. 11A) or 50 mg/kg (FIG. 11B).

FIGS. 12A-D show the half-life determination of SABA1-SABA4 in mice.

FIG. 13A shows a graph summary of half-life enhancement in mice ofSABA1-4 when co-injected with HSA. FIG. 13 b compares data fromcynomolgus monkey and mice.

FIGS. 14A-B show the half-life determination for SABA1.1 and SABA5.1 incynomolgus monkey.

FIG. 15 shows SABA1.2 binding to albumins from human, mouse and rat bydirect binding ELISA assay.

FIG. 16 shows the determination of SABA1.1 and HSA stoichiometry.

FIG. 17 shows Biacore analysis of SABA1.2 binding to recombinant domainfragments of HSA.

FIG. 18 shows the pharmacokinetic profile for SABA1.2 in monkeys dosedat 1 mpk and 10 mpk.

FIG. 19 shows the pharmacokinetic profile for SABA1.2 in monkeys dosedintravenously or subcutaneously at 1 mpk.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D). Thepeptides of the invention are proteins derived from the tenth type IIIdomain of fibronectin that have been modified to bind specifically tothe p19 subunit of IL-23 and are referred to herein as “Adnectin” or“anti-IL-23 Adnectin”.

The term “PK” is an acronym for “pharmokinetic” and encompassesproperties of a compound including, by way of example, absorption,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” refers to any protein, peptide, or moiety thataffects the pharmacokinetic properties of a biologically active moleculewhen fused to or administered together with the biologically activemolecule. Examples of a PK modulation protein or PK moiety include PEG,human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos.2005/0287153 and 2007/0003549), human serum albumin, Fc or Fc fragments,and sugars (e.g., sialic acid).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing condition using Coomassie blue or, preferably, silver stain.Isolated polypeptide includes the polypeptide in situ within recombinantcells since at least one component of the polypeptide's naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The “half-life” of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thesequence or compound and/or clearance or sequestration of the sequenceor compound by natural mechanisms. The half-life can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to theprimate a suitable dose of the amino acid sequence or compound of theinvention; collecting blood samples or other samples from said primateat regular intervals; determining the level or concentration of theamino acid sequence or compound of the invention in said blood sample;and calculating, from (a plot of) the data thus obtained, the time untilthe level or concentration of the amino acid sequence or compound of theinvention has been reduced by 50% compared to the initial level upondosing. Reference is for example made to the standard handbooks, such asKenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Lee, P. I. D. et al., Pharmacokinetic Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker, publ. (1982).

Half-life can be expressed using parameters such as the t1/2-alpha,t1/2-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, any two of these parameters, or all three theseparameters. An “increase in half-life” in particular refers to anincrease in the t1/2-beta, either with or without an increase in thet1/2-alpha and/or the AUC or both.

Overview

The application provides Adnectins against human IL-23-specific p19subunit. In order to identify IL-23 specific antagonist, IL-23 waspresented to large synthetic libraries of Adnectin using anti-p40 mAbs.Adnectins that bound to IL-23 p19 subunit were screened for binding tohuman IL-23, competition of the IL-23/IL-23R interaction and inhibitionof IL-23 induced signaling in a T-cell line. The anti-IL-23 Adnectinswere subjected to further selective pressure by lowering the targetconcentration and selecting for anti-IL-23 Adnectins with slowoff-rates. From this optimization process a family of Adnectins wereidentified as IL-23 specific inhibitors with favorable biochemical andbiophysical properties.

Fibronectin Based Scaffolds

One aspect of the application provides for polypeptides comprising Fn3domain in which one or more of the solvent accessible loops has beenrandomized or mutated. In some embodiments, the Fn3 domain is an Fn3domain derived from the wild-type tenth module of the human fibronectintype III domain(¹⁰Fn3):VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1). In the ¹⁰Fn3sequence above, the BC, DE and FG loops are underlined.

A variety of mutant ¹⁰Fn3 scaffolds have been reported. In one aspect,one or more of Asp 7, Glu 9, and Asp 23 is replaced by another aminoacid, such as, for example, a non-negatively charged amino acid residue(e.g., Asn, Lys, etc.). These mutations have been reported to have theeffect of promoting greater stability of the mutant ¹⁰Fn3 at neutral pHas compared to the wild-type form (See, PCT Publication No. WO02/04523).A variety of additional alterations in the ¹⁰Fn3 scaffold that areeither beneficial or neutral have been disclosed. See, for example,Batori et al., Protein Eng., 15(12):1015-1020 (December 2002); Koide etal., Biochemistry, 40(34):10326-10333 (Aug. 28, 2001).

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO:1, the AB loopcorresponds to residues 15-16, the BC loop corresponds to residues21-30, the CD loop corresponds to residues 39-45, the DE loopcorresponds to residues 51-56, the EF loop corresponds to residues60-66, and the FG loop corresponds to residues 76-87 (Xu et al.,Chemistry & Biology, 9:933-942 (2002)).

In some embodiments, the ¹⁰Fn3 polypeptide may be at least 40%, 50%,60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3domain, shown in SEQ ID NO:1. Much of the variability will generallyoccur in one or more of the loops. Each of the beta or beta-like strandsof a ¹⁰Fn3 polypeptide may consist essentially of an amino acid sequencethat is at least 80%, 85%, 90%, 95% or 100% identical to the sequence ofa corresponding beta or beta-like strand of SEQ ID NO:1, provided thatsuch variation does not disrupt the stability of the polypeptide inphysiological conditions.

In some embodiments, the disclosure provides polypeptides comprising atenth fibronectin type III (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domaincomprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; anda loop FG; and has at least one loop selected from loop BC, DE, and FGwith an altered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain. In some embodiments, theBC and FG loops are altered, in some embodiments, the BC, DE, and FGloops are altered, i.e., the Fn3 domains comprise non-naturallyoccurring loops. By “altered” is meant one or more amino acid sequencealterations relative to a template sequence (corresponding humanfibronectin domain) and includes amino acid additions, deletions, andsubstitutions. Altering an amino acid sequence may be accomplishedthrough intentional, blind, or spontaneous sequence variation, generallyof a nucleic acid coding sequence, and may occur by any technique, forexample, PCR, error-prone PCR, or chemical DNA synthesis.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In some embodiments, the length of the loop may beextended by 2-25 amino acids. In some embodiments, the length of theloop may be decreased by 1-11 amino acids. To optimize antigen binding,therefore, the length of a loop of ¹⁰Fn3 may be altered in length aswell as in sequence to obtain the greatest possible flexibility andaffinity in antigen binding.

In some embodiments, the polypeptide comprises a Fn3 domain thatcomprises an amino acid sequence at least 80, 85, 90, 95, 98, 99 or 100%identical to the non-loop regions of SEQ ID NO:1, wherein at least oneloop selected from BC, DE, and FG is altered. In some embodiments, thealtered BC loop has up to 10 amino acid substitutions, up to 4 aminoacid deletions, up to 10 amino acid insertions, or a combinationthereof. In some embodiments, the altered DE loop has up to 6 amino acidsubstitutions, up to 4 amino acid deletions, up to 13 amino acidinsertions or a combination thereof. In some embodiments, the FG loophas up to 12 amino acid substitutions, up to 11 amino acid deletions, upto 25 amino acid insertions or a combination thereof.

In some embodiments, the BC loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofGHYPMHV (SEQ ID NO: 2), GHYPLHV (SEQ ID NO: 3), GHYPMHI (SEQ ID NO:4),GHYPLHI (SEQ ID NO:5) and GHYPLHL (SEQ ID NO:6).

In some embodiments, the DE loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofHRTH(SEQ ID NO:7), YYHY(SEQ ID NO:8), SKQH (SEQ ID NO:9), SNVH (SEQ IDNO:10), NRAH (SEQ ID NO:11), RKTY(SEQ ID NO:12), RSRY (SEQ ID NO:13),SRYY (SEQ ID NO:14), PHRY (SEQ ID NO:15), RSTH (SEQ ID NO:16), SRIY (SEQID NO:17), HQRY (SEQ ID NO:18), KQVY (SEQ ID NO:19), AHRY (SEQ IDNO:20), RSRH (SEQ ID NO:21), ARQY (SEQ ID NO:22), RTQY (SEQ ID NO:23),PRYH (SEQ ID NO:24), MRQH (SEQ ID NO:25), SRKY (SEQ ID NO:26), RQKY (SEQID NO:27), HAKY(SEQ ID NO:28), SNRY (SEQ ID NO:29), NTSH (SEQ ID NO:30),SQVY (SEQ ID NO:31), NRVY (SEQ ID NO:32), PRSH (SEQ ID NO:33), RTKY (SEQID NO:34), SRYH (SEQ ID NO:35), PRRY (SEQ ID NO:36), RQKY (SEQ IDNO:37), RYKY (SEQ ID NO:38), VPRH (SEQ ID NO:39), TPKH (SEQ ID NO:40),RSKY (SEQ ID NO:41), SRKY (SEQ ID NO:42), VPRY (SEQ ID NO:43), PRRY (SEQID NO:44), RMRH (SEQ ID NO:45), PPRH (SEQ ID NO:46), RQIY (SEQ IDNO:47), and MRQH (SEQ ID NO:48).

In some embodiments, the FG loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofYYNEADYSQI (SEQ ID NO:49), YYQEYEYRYI (SEQ ID NO:50), YYMEEKYAVI (SEQ IDNO:51), YYAQENYKEI (SEQ ID NO:52), YYKEANYREI (SEQ ID NO:53), YYAQEEYHII(SEQ ID NO:54), YYKEADYSQI (SEQ ID NO:55), YYEQVEYREI (SEQ ID NO:56),YYEQPIYATI (SEQ ID NO:57), YYEQVEYREI (SEQ ID NO:58) and YYSEELYKYI (SEQID NO:59).

The ¹⁰Fn3 domain may begin with amino acid alterations. For example, anadditional MG sequence may be placed at the N-terminus of an Fn3 domain.The M will usually be cleaved off, leaving a G at the N-terminus In someembodiments, sequences may be placed at the C-terminus of the ¹⁰Fn3domain. For example, in site directed PEGylation where a cysteinecontaining linker such as GSGC (SEQ ID NO: 101) is added to theC-terminus. Alternatively, PEGylation of the naturally occurringC-terminus tail that has been mutated by changing the Ser to a Cys for acysteine containing linker EIDKPCQ (SEQ ID NO: 102). Examples of theanti-IL-23 adnectin of the invention comprising the GSGC linker includeATI001014, ATI001015, ATI001016, ATI001044, ATI001045 and ATI001047.ATI000934 is an example of the anti-11-23 adnectin of the inventioncomprising the EIDKPCQ linker.

In some embodiments, the protein of the invention comprises one loopsequence from the BC loop sequences shown in SEQ ID NOs: 2-6, one DEloop sequence shown in SEQ ID NOs: 7-48 and one FG loop sequence shownin SEQ ID NOs: 49-59. In some embodiments, the protein of the inventioncomprises a BC, DE and FG loop amino acid sequence at least 70, 75, 80,85, 90, 95, 98, 99 or 100% identical to of any one of SEQ ID NOS:2-59.

Further, one skilled in the art will recognize that BC loop sequencesshown in SEQ ID NO: 2-6 share a common sequence motif GHYPX₁HX₂ (SEQ IDNO:257) where X₁ is either M or L, and X₂ is either I or V, and the FGloop sequences shown in SEQ ID NO: 49-59 share a common sequence motifYYX₃X₃X₃X₃YX₃X₃I (SEQ ID NO: 258) where X₃ can be any amino acid. Itwould therefore be possible to generate additional Adnectins that bindIL-23 with BC loops that fit the consensus sequence GHYPX₁HX₂ and/orwith other FG loops, beyond those explicitly listed in SEQ ID NOS:49-59,that fit the pattern YYX₃X₃X₃X₃YX₃X₃I.

In some embodiments, the anti-IL-23 Adnectin comprises the amino acidsequence of any one of SEQ ID NOS:60-100. In some embodiments, theanti-IL-23 Adnectin comprises the Fn3 domain amino acid sequence fromposition 3-96 of any one of SEQ ID NOS:60-100. In some embodiments, theanti-IL-23 Adnectin comprises the amino acid sequence at least 70, 75,80, 85, 90, 95, 98, 99 or 100% identical to any one of SEQ ID NOS:60-100. In some embodiments, the anti-IL-23 Adnectin comprises the aminoacid sequence at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% identicalto amino acid sequence from position 3-96 any one of SEQ ID NOS:60-100.

In some embodiments, the anti-IL-23 Adnectin may be pegylated and/orcontain a his-tag. As used herein, ATI000934 refers to a protein whereinthe loop sequences are identical to those of construct 1571G06 (Seq ID87), and the protein contains the residues EIDKPCQ at the C-terminuswhere the protein is pegylated and contains a his-tag. ATI001014 refersto a protein wherein the loop sequences are identical to those ofconstruct 1571G04 (Seq ID 86), and the protein contains a GSGC linker atthe C-terminus where the protein is pegylated and contains a his-tag.ATI001015 refers to a protein wherein the loop sequences are identicalto those of construct 1572G06 (Seq ID 91), and the protein contains aGSGC linker at the C-terminus where the protein is pegylated andcontains a his-tag. ATI001016 refers to a protein wherein the loopsequences are identical to those of construct 1490B03 (Seq ID 79), andthe protein contains a GSGC linker at the C-terminus where the proteinis pegylated and contains a his-tag. ATI001044 refers to a proteinwherein the loop sequences are identical to those of construct1490B03(Seq ID 79), and the protein contains a GSGC linker at the C-terminus,but protein is not pegylated and there is no his tag. ATI001045 refersto a protein wherein the loop sequences are identical to those ofconstruct 1490B03 (Seq ID 79), and the protein contains a GSGC linker atthe C-terminus where the protein is pegylated; and there is no his tag.ATI001047 refers to a protein wherein the loop sequences are identicalto those of construct 1571G04 (Seq ID 86), and the protein contains aGSGC linker at the C-terminus where the protein is pegylated, and thereis no his tag

Fibronectin naturally binds certain types of integrins through itsintegrin-binding motif, “arginine-glycine˜aspartic acid” (RGD). In someembodiments, the polypeptide comprises a ¹⁰Fn3 domain that lacks the(RGD) integrin binding motif

Pharmacokinetic Moieties

In one aspect, the application provides for anti-IL-23 Adnectin furthercomprising a pharmacokinetic (PK) moiety. Improved pharmacokinetics maybe assessed according to the perceived therapeutic need. Often it isdesirable to increase bioavailability and/or increase the time betweendoses, possibly by increasing the time that a protein remains availablein the serum after dosing. In some instances, it is desirable to improvethe continuity of the serum concentration of the protein over time(e.g., decrease the difference in serum concentration of the proteinshortly after administration and shortly before the nextadministration). The anti-IL-23 Adnectin may be attached to a moietythat reduces the clearance rate of the polypeptide in a mammal (e.g.,mouse, rat, or human) by greater than three-fold relative to theunmodified Adnectin. Other measures of improved pharmacokinetics mayinclude serum half-life, which is often divided into an alpha phase anda beta phase. Either or both phases may be improved significantly byaddition of an appropriate moiety.

Moieties that tend to slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties, e.g.,polyethylene glycol, sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc, Fc fragments, transferrin, or serum albumin)The Adnectin may be fused to albumin or a fragment (portion) or variantof albumin as described in U.S. Publication No. 2007/0048282.

In some embodiments, the PK moiety is a serum albumin binding proteinsuch as those described in U.S. Publication Nos. 2007/0178082 and2007/0269422.

In some embodiments, the PK moiety is a serum immunoglobulin bindingprotein such as those described in U.S. Publication No. 2007/0178082.

In some embodiments, the Adnectin comprises polyethylene glycol (PEG).One or more PEG molecules may be attached at different positions on theprotein, and such attachment may be achieved by reaction with amines,thiols or other suitable reactive groups. The amine moiety may be, forexample, a primary amine found at the N-terminus of a polypeptide or anamine group present in an amino acid, such as lysine or arginine. Insome embodiments, the PEG moiety is attached at a position on thepolypeptide selected from the group consisting of: a) the N-terminus; b)between the N-terminus and the most N-terminal beta strand or beta-likestrand; c) a loop positioned on a face of the polypeptide opposite thetarget-binding site; d) between the C-terminus and the most C-terminalbeta strand or beta-like strand; and e) at the C-terminus.

Pegylation may be achieved by site-directed pegylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere pegylation preferentially occurs. In some embodiments, the proteinis modified to introduce a cysteine residue at a desired position,permitting site directed pegylation on the cysteine. PEG may vary widelyin molecular weight and may be branched or linear.

In some embodiments, the Adnectin comprises an Fn3 domain and a PKmoiety. In some embodiments, the Fn3 domain is a ¹⁰Fn3 domain. In someembodiments, the PK moiety increases the serum half-life of thepolypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domainalone.

In some embodiments, the PK moiety is a polymeric sugar. In someembodiments, the PK moiety is a polyethylene glycol moiety. In someembodiments the PK moiety is a serum albumin binding protein. In someembodiments the PK moiety is human serum albumin. In some embodimentsthe PK moiety is a serum immunoglobulin binding protein. In someembodiments, the PK moiety is transferrin. In some embodiments the PKmoiety is another Adnectin specific for a serum protein.

Biophysical and Biochemical Characterization

The application provides Adnectin comprising a Fn3 domain that binds tothe p19 subunit of IL-23. As shown in Table 1 and Example 4, polypeptidebinding to a target molecule may be assessed in terms of equilibriumconstants (e.g., dissociation, KO and in terms of kinetic constants(e.g., on-rate constant, K. and off-rate constant, k_(off)). An Adnectinwill generally bind to a target molecule with a K_(D) of less than 500nM, 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, although higher K_(D)values may be tolerated where the K_(off) is sufficiently low or theK_(on), is sufficiently high.

The BC, DE and FG loop sequences of the family of anti-IL-23 Adnectin ofthe invention are presented in Table 1 below, as well as thecorresponding full length SEQ ID NO.

TABLE 1 Anti-IL-23 Adnectin Family On-rate Off-rate Affinity Kit 225 SEQClone BC DE (k_(a), (k_(d), (K_(D), pSTAT3 ID ID Loop Loop FG loopM⁻¹s⁻¹) s⁻¹) M) IC50 (nM) NO 1434A08 GHYPMHV HRTH YYNEADYSQI n.d. n.d.n.d.  4.9  60 1437G04 GHYPLHV YYHY YYNEADYSQI 1.34E+05 4.46E−04 3.3E−09 3.3  61 1437A09 GHYPMHV SKQH YYNEADYSQI 8.06E+04 4.60E−04 5.7E−09 20.2 62 1438E05 GHYPMHV SNVH YYNEADYSQI n.d. n.d. n.d. 39.6  63 1438D01GHYPMHV NRAH YYNEADYSQI n.d. n.d. n.d. 11.4  64 1438B02 GHYPMHV RKTYYYNEADYSQI n.d. n.d. n.d.  8.6  65 1438A09 GHYPMHV RSRY YYNEADYSQI n.d.n.d. n.d. 10.8  66 1486G03 GHYPMHV SRYY YYNEADYSQI 8.62E+04 4.25E−044.9E−09  3  67 1486C04 GHYPMHV PHRY YYNEADYSQI 1.12E+05 3.79E−04 3.4E−0931.9  68 1486D04 GHYPLHI RSTH YYNEADYSQI 1.51E+05 3.52E−04 2.3E−09  2 69 1486B05 GHYPMHV SRIY YYNEADYSQI 1.34E+05 3.81E−04 2.8E−09  4  701486D05 GHYPLHV HQRY YYNEADYSQI 1.20E+05 3.44E−04 2.9E−09  4  71 1487C03GHYPLHI KQVY YYNEADYSQI 1.61E+05 3.82E−04 2.4E−09  3.7  72 1487G03GHYPLHV AHRY YYNEADYSQI 1.03E+05 3.16E−04 3.1E−09  3  73 1487D09 GHYPMHIRSRH YYNEADYSQI 1.50E+05 2.50E−04 1.7E−09  2  74 1487H04 GHYPMHV ARQYYYQEYEYRYI 5.96E+04 too slow <nM  2  75 to measure 1490 E02 GHYPMHV RTQYYYNEADYSQI 9.73E+04 4.65E−04 4.8E−09 n.d.  76 1490G02 GHYPMHV PRYHYYMEEKYAVI 1.97E+05 3.32E−04 1.7E−09  0.4  77 1490H05 GHYPLHV MRQHYYAQENYKEI 1.56E+05 3.22E−04 2.1E−09  0.4  78 1490B03 GHYPLHV SRKYYYKEANYREI 1.53E+05 too slow <nM  0.3  79 to measure 1490H06 GHYPLHIRQKY YYNEADYSQI 9.35E+04 too slow <nM  2.3  80 to measure 1490A07GHYPLHI HAKY YYAQENYKEI 1.63E+05 too slow <nM  0.6  81 to measure1490C07 GHYPLHV SNRY YYNEADYSQI 9.56E+04 4.26E−04 4.5E−09  5.6  821490H08 GHYPLHI NTSH YYNEADYSQI 1.67E+05 5.87E−04 3.5E−09  83 1491A05GHYPLHV SQVY YYAQENYKEI 1.98E+05 4.08E−04 2.1E−09  0.3  84 1571H03GHYPLHV NRVY YYAQEEYHII 1.18E+05 3.95E−04 3.4E−09  0.4  85 1571G04GHYPLHV PRSH YYAQENYKEI 1.70E+05 3.61E−04 2.1E−09  0.1  86 1571G06GHYPLHL RTKY YYKEADYSQI 1.31E+05 2.38E−04 1.8133E−09  0.3  87 1571F10GHYPLHI SRYH YYEQVEYREI 2.68E+05 too slow <nM  0.1  88 to measure1572D04 GHYPMHV PRRY YYEQPIYATI 1.03E+05 2.94E−04 2.9E−09  4  89 1572F05GHYPLHI RQKY YYNEADYSQI 1.32E+05 2.85E−04 2.2E−09  3  90 1572G06 GHYPLHVRYKY YYAQENYKEI 1.05E+05 too slow <nM  0.05  91 to measure 1572B10GHYPMHV VPRH YYNEADYSQI 6.53E+04 too slow <nM  0.8  92 to measure1572C09 GHYPMHI TPKH YYNEADYSQI 6.07E+04 too slow <nM  0.5  93to measure 1572H05 GHYPLHI RSKY YYEQVEYREI 3.75E+05 2.30E−04 6.1E−10 0.01  94 1572H08 GHYPLHV SRKY YYNEADYSQI 5.73E+04 too slow <nM  2.8  95to measure 1550A07 GHYPMHV VPRY YYAQENYKEI 1.17E+05 1.33E−04 1.1E−09 1.0  96 1550C05 GHYPMHV PRRY YYNEADYSQI 6.59E+04 1.35E−04 2.1E−09 16.8 97 1550 E03 GHYPLHI RMRH YYSEELYKYI 9.95E+04 1.86E−04 1.9E−09  5.3  981550 E06 GHYPMHV PPRH YYAQENYKEI 5.54E+04 1.16E−04 2.1E−09  0.4  991550H05 GHYPLHV RQIY YYNEADYSQI 6.52E+04 2.12E−04 3.3E−09  1.5 100*Method for affinity determinations: The anti-His antibody, mAb050 (RnDSystems, MN) was diluted to 20 ug/mL in acetate 5.0 and immobilized to~9000 RU on flow cells 1 and 2 of a CM5 chip surface (GE Healthcare,Piscataway, NJ) according to the manufacturer's instructions. Allsurface plasmon experiments were conducted in HBS-EP (10 mM Hepes 150 mMNaCl 3 mM EDTA 0.05% Surfactant P20) at 25° C. IL-23 was injected overanti-His mAb captured Adnectins for 2 minutes followed by a 10 minutedissociation phase. Evaluation of the binding specificity was completedusing Biacore T100 evaluation software. Additional detailed methods aredescribed in Example 4.

Additional anti-IL-23 Adnectin characterization is described in Table 2.

TABLE 2 Anti-IL-23 Adnectin IC50/EC50 PBMNC clone pSTAT3 IL-17 EC50IL-22 EC50 ID BC DE FG IC50 (nM) (nM) (nM) 1571G04 GHYPLHV PRSHYYAQENYKEI 0.23 ± .05 1.4 ± 0.3 1.3 ± 0.7 1490B03 GHYPLHV SRKYYYKEANYREI  .09 ± .01 1.4 ± 0.1 1.7 ± 0.8 1572G06 GHYPLHV RYKYYYAQENYKEI  .21 ± .03 1.6 ± 0.3 1.9 ± 0.3 1550E06 GHYPMHV PPRHYYAQENYKEI 1.15 ± .5 1.5 ± 0.6 2.1 ± 0.8 1571H03 GHYPLHV NRVY YYAQEEYHIIn.d. 2.9 ± 0.8 2.3 ± 0.4 1490H05 GHYPLHV MRQH YYAQENYKEI n.d. 1.8 ± 2.12.9 ± 1.0 1571G06 GHYPLHL RTKY YYKEADYSQI 0.93 ± .5 3.5 ± 1.4 5.1 ± 4.31572C09 GHYPMHI TPKH YYNEADYSQI n.d. 7.9 ± 6.1 5.3 ± 4.5 (n.d. notdetermined) (Detailed methods described in Example 4).Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides Adnectin comprising fibronectintype III domains that bind p19 subunit of IL-23. One way to rapidly makeand test Fn3 domains with specific binding properties is the nucleicacid-protein fusion technology of Adnexus, a Bristol-Myers Squibb R&DCompany. This disclosure utilizes the in vitro expression and taggingtechnology, termed PROfusion, which exploits nucleic acid-proteinfusions (RNA- and DNA-protein fusions) to identify novel polypeptidesand amino acid motifs that are important for binding to proteins.Nucleic acid-protein fusion technology is a technology that covalentlycouples a protein to its encoding genetic information. For a detaileddescription of the RNA-protein fusion technology and fibronectin-basedscaffold protein library screening methods see .Szostak et al., U.S.Pat. Nos. 6,258,558, 6,261,804, 6,214,553, 6,281,344, 6,207,446,6,518,018, 6,818,418; and Roberts et al., Proc. Natl., Acad. Sci.,94:12297-12302 (1997), herein incorporated by reference.

Vectors and Polynucleotides Embodiments

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21,2003); Sinclair et al., Protein Expr. Purif., 26(I):96-105 (October2002); Connell, N. D., Curr. Opin. Biotechnol., 12(5):446-449 (October2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (September 1996);and Sharp et al., Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press, publ. (1989),or Ausubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York, publ. (1987) and periodicupdates, herein incorporated by reference. Generally, the DNA encodingthe polypeptide is operably linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, viral, orinsect genes. Such regulatory elements include a transcriptionalpromoter, an optional operator sequence to control transcription, asequence encoding suitable mRNA ribosomal binding sites, and sequencesthat control the termination of transcription and translation. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene, to facilitate recognition oftransformants, are additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in U.S. Pat. No. 5,631,144. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available. The DNA for suchprecursor regions may be ligated in reading frame to DNA encoding theprotein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein of the invention, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein of the invention. Promoter sequences are also knownfor eukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tall to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding proteins of the invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, .alpha.-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepeptide-encoding sequence, but is preferably located at a site 5′ fromthe promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein of the invention.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO 94/11026 and the expressionvector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, Elsevier, New York, publ.(1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces genus, such as S. cerevisiae, may alsobe used for production of polypeptides. Various mammalian or insect cellculture systems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow et al. (Bio/Technology, 6:47 (1988)).Examples of suitable mammalian host cell lines include endothelialcells, CO8-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.In the examples shown here, the host cells used for high-throughputprotein production (HTPP) and mid-scale production was the BL21 DE3plysS-bacterial strain. The host cells used to produce the proteins ofthis invention may be cultured in a variety of media, such as thosedescribed in Ham et al., Meth. Enzymol., 58:44 (1979), Barites et al.,Anal. Biochem., 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866,4,927,762, 4,560,655, 5,122,469, 6,048,728, 5,672,502, or RE30,985. Anyother necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system).

Proteins of the invention can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2ndEdition, The Pierce Chemical Co., Rockford, Ill., publ. (1984).Modifications to the protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, or preferablyat least 95% pure, and most preferably at least 98% pure. Regardless ofthe exact numerical value of the purity, the polypeptide is sufficientlypure for use as a pharmaceutical product.

A platform manufacturing process was used to prepare anti-IL-23Adnectin. Example 1 describes an example of the manufacturing process.The Adnectin is produced in Escherichia coli (E. coli). E. coli MG1655cells were transformed with expression vector (pBMS2008/ATI001044) whichproduces the protein in an insoluble form as inclusion bodies. Therecombinant strain is grown in stirred tank fermentors. At the end offermentation the inclusion bodies are collected, solubilized, andrefolded in preparation for purification. The purified Adnectin isconjugated to a 40 kDa branched methoxyPEG using a maleimide linker. Theconjugated material is subsequently repurified to remove free PEG, freeAdnectin and product related impurities. Quality control testing isperformed on the bulk drug substance.

Therapeutic In Vivo Uses

In one aspect, the application provides anti-IL-23 Adnectin useful inthe treatment of autoimmune diseases such as lupus (e.g., lupuserythematosus, lupus nephritis), Hashimoto's thyroiditis, primarymyxedema, Graves' disease, pernicious anemia, autoimmune atrophicgastritis, Addison's disease, diabetes (e.g., insulin dependent diabetesmellitis, type I diabetes mellitis), Goodpasture's syndrome, myastheniagravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmuneuveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathicthrombocytopenia, primary biliary cirrhosis, chronic action hepatitis,ulceratis colitis, Sjögren's syndrome, rheumatic diseases (e.g.,rheumatoid arthritis), polymyositis, scleroderma, and mixed connectivetissue disease.

The application also provides methods for administering anti-IL-23Adnectins to a subject. In some embodiments, the subject is a human. Insome embodiments, the anti-IL-23 Adnectins are pharmaceuticallyacceptable to a mammal, in particular a human. A “pharmaceuticallyacceptable” polypeptide refers to a polypeptide that is administered toan animal without significant adverse medical consequences, such asessentially endotoxin free or having very low endotoxin levels.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the anti-IL-23 Adnectin described herein,wherein the composition is essentially endotoxin free. Therapeuticformulations comprising anti-IL-23 Adnectin are prepared for storage bymixing the described Adnectin having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Osol, A., ed., Remington's Pharmaceutical Sciences, 16th Edition(1980)), in the form of aqueous solutions, lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethoninm chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as Tween, PLURONIC® or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The skilled artisan will understand that the dosage of each therapeuticagent will be dependent on the identity of the agent.

For therapeutic applications, the anti-IL-23 Adnectin is administered toa subject, in a pharmaceutically acceptable dosage form. It can beadministered intravenously as a bolus or by continuous infusion over aperiod of time, or by subcutaneous routes. Suitable pharmaceuticallyacceptable carriers, diluents, and excipients are well known and can bedetermined by those of skill in the art as the clinical situationwarrants. Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, (2) 0.9% saline (0.9%w/v NaCl), and (3) 5% (w/v) dextrose.

The method of the present invention can be practiced in vitro, in vivo,or ex vivo.

Administration of anti-IL-23 Adnectin, and one or more additionaltherapeutic agents, whether co-administered or administeredsequentially, may occur as described above for therapeutic applications.Suitable pharmaceutically acceptable carriers, diluents, and excipientsfor co-administration will be understood by the skilled artisan todepend on the identity of the particular therapeutic agent beingadministered.

When present in an aqueous dosage form, rather than being lyophilized,the protein typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted. For the treatment of disease, the appropriate dosage ofanti-IL-23 Adnectin will depend on the type of disease to be treated,the severity and course of the disease, whether the Adnectin isadministered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to theAdnectin, and the discretion of the attending physician. The protein issuitably administered to the patient at one time or over a series oftreatments.

Fusions of Serum Albumin Binding Adnectin (SABA)

In certain aspects, the application provides fusion proteins comprisinganti-IL23-Adnectin fused to a ¹⁰Fn3 domains that binds to human serumalbumin (a Serum Albumin Binding Adnectin (¹⁰Fn3 domain) or SABA). Suchfusion proteins have extended serum half lives in the presence ofalbumin relative to anti-IL23-Adnectin alone.

In certain aspects, the application provides fusion proteins comprising¹⁰Fn3 domains that bind specifically to serum albumin, e.g., human serumalbumin (HSA) to prolong the t_(1/2) of the fusion protein.

In certain embodiments, the serum half-life of the anti-IL23-Adnectinfused to the SABA is increased relative to the serum half-life of theanti-IL23-Adnectin when not conjugated to the SABA. In certainembodiments, the serum half-life of the SABA fusion is at least 20, 40,60, 80, 100, 120, 150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800,1900, 2000, 2500, or 3000% longer relative to the serum half-life of theanti-IL23-Adnectin when not fused to the SABA. In other embodiments, theserum half-life of the SABA fusion is at least 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold,8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold,25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than theserum half-life of the anti-IL23-Adnectin when not fused to the SABA. Insome embodiments, the serum half-life of the SABA fusion is at least 10hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200hours.

Accordingly, the SABA fusion molecules described herein are useful forincreasing the half-life of anti-IL23-Adnectin by creating a fusionbetween anti-IL23-Adnectin and the SABA. Such fusion molecules may beused to treat conditions which respond to the biological activity ofIL23. The present invention contemplates the use of the SABA fusionmolecules in diseases caused by the disregulation of IL-23.

The fusion may be formed by attaching anti-IL23-Adnectin to either endof the SABA molecule, i.e., SABA-anti-IL23-Adnectin oranti-IL23-Adnectin-SABA arrangements.

In one aspect, the disclosure provides fusion proteins comprisinganti-IL23-Adnectin comprising a serum albumin binding ¹⁰Fn3 domain. Inexemplary embodiments, the serum albumin binding Fn3 proteins describedherein bind to HSA with a K_(D) of less than 3 uM, 2.5 uM, 2 uM, 1.5 uM,1 uM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 200 pM. 100 pM, 50 pMor 10 pM. In certain embodiments, the serum albumin binding ¹⁰Fn3proteins described herein bind to HSA with a K_(D) of less than 3 uM,2.5 uM, 2 uM, 1.5 uM, 1 uM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM,200 pM. 100 pM, 50 pM or 10 pM at a pH range of 5.5 to 7.4 at 25° C. or37° C. In some embodiments, the serum albumin binding ¹⁰Fn3 proteinsdescribed herein bind more tightly to HSA at a pH less than 7.4 ascompared to the binding affinity for HSA at a pH of 7.4 or greater.

In certain embodiments, the fusion proteins comprising HSA binding ¹⁰Fn3domains described herein may also bind serum albumin from one or more ofmonkey, rat, or mouse. In certain embodiments, the serum albumin bindingFn3 proteins described herein bind to rhesus serum albumin (RhSA) orcynomolgus monkey serum albumin (CySA) with a K_(D) of less than 3 uM,2.5 uM, 2 uM, 1.5 uM, 1 uM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pMor 100 pM.

In certain embodiments, the fusion proteins comprising serum albuminbinding ¹⁰Fn3 domains described herein bind to domain I and/or domain IIof HSA. In one embodiment, the fusion proteins comprising serum albuminbinding Fn3 domains described herein do not bind to domain III of HSA.

In certain embodiments, the serum albumin binding ¹⁰Fn3 (SABA) portionof the fusion proteins comprises a sequence having at least 40%, 50%,60%, 70%, 75%, 80% or 85% identity to the wild-type ¹⁰Fn3 domain (SEQ IDNO: 1). In one embodiment, at least one of the BC, DE, or FG loops ismodified relative to the wild-type ¹⁰Fn3 domain. In another embodiment,at least two of the BC, DE, or FG loops are modified relative to thewild-type ¹⁰Fn3 domain. In another embodiment, all three of the BC, DE,and FG loops are modified relative to the wild-type ¹⁰Fn3 domain. Inother embodiments, a SABA comprises a sequence having at least 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any one of the 26 coreSABA sequences shown in Table 3 (i.e., SEQ ID NO: 103, 107, 111, 115,119, and 123-143) or any one of the extended SABA sequences shown inTable 3 (i.e., SEQ ID NO: 188-215, minus the 6×HIS tag).

In certain embodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues. In exemplaryembodiments, the BC, DE, and FG loops are replaced with polypeptidescomprising the BC, DE and FG loop sequences from any of the HSA bindersshown in Table 3 below (i.e., SEQ ID NOs: 103, 107, 111, 115, 119, and123-143 in Table 3).

In certain embodiments, a SABA (e.g., a SABA core sequence or a sequencebased thereon as described above) may be modified to comprise anN-terminal extension sequence and/or a C-terminal extension sequence.Exemplary extension sequences are shown in Table 3. For example, SEQ IDNO: 188 designated as SABA1.1 comprises the core SABA 1 sequence (SEQ IDNO: 103) with an N-terminal sequence MGVSDVPRDLE (SEQ ID NO: 144,designated as AdNT1), and a C-terminal sequence EIDKPSQ (SEQ ID NO:153). SABA1.1 further comprises a His6 tag at the C-terminus, however,it should be understood that the His6 tag is completely optional and maybe placed anywhere within the N- or C-terminal extension sequences.Further, any of the exemplary N- or C-terminal extension sequencesprovided in Table 3 (SEQ ID NO: 144-163), and any variants thereof, canbe used to modify any given SABA core sequence provided in Table 3.

In other embodiments, the tail sequences may be combined with otherknown linker sequences (e.g., SEQ ID NO: 164-187 in Table 3) asnecessary when designing a SABA fusion molecule.

Conjugation Linkers

SABA fusions may be covalently or non-covalently linked. In someembodiments, a serum albumin binding ¹⁰Fn3 may be directly or indirectlylinked to a anti-IL23-Adnectin via a polypeptide linker. Suitablelinkers for joining Fn3 are those which allow the separate domains tofold independently of each other forming a three dimensional structurethat permits high affinity binding to a target molecule.

The disclosure provides a number of suitable linkers that meet theserequirements, including glycine-serine based linkers, glycine-prolinebased linkers, as well as the linker having the amino acid sequencePSTSTST (SEQ ID NO: 184). The Examples described herein demonstrate thatFn3 domains joined via polypeptide linkers retain their target bindingfunction. In some embodiments, the linker is a glycine-serine basedlinker. These linkers comprise glycine and serine residues and may bebetween 8 and 50, 10 and 30, and 10 and 20 amino acids in length.Examples include linkers having an amino acid sequence (GS)₇ (SEQ ID NO:171), G(GS)₆ (SEQ ID NO: 166), and G(GS)₇G (SEQ ID NO: 168). Otherlinkers contain glutamic acid, and include, for example, (GSE)₅ (SEQ IDNO: 173) and GGSE GGSE (SEQ ID NO: 177). Other exemplary glycine-serinelinkers include (GS)₄ (SEQ ID NO: 170), (GGGGS)₇ (SEQ ID NO: 179),(GGGGS)₅ (SEQ ID NO: 180), and (GGGGS)₃G (SEQ ID NO: 181). In someembodiments, the linker is a glycine-proline based linker. These linkerscomprise glycine and proline residues and may be between 3 and 30, 10and 30, and 3 and 20 amino acids in length. Examples include linkershaving an amino acid sequence (GP)₃G (SEQ ID NO: 182) and (GP)₅G (SEQ IDNO: 183). In other embodiments, the linker may be a proline-alaninebased linker having between 3 and 30, 10 and 30, and 3 and 20 aminoacids in length. Examples of proline alanine based linkers include, forexample, (PA)₃ (SEQ ID NO: 185), (PA)₆ (SEQ ID NO: 186) and (PA)₉ (SEQID NO: 187). It is contemplated, that the optimal linker length andamino acid composition may be determined by routine experimentation bymethods well known in the art.

In some embodiments, the fusions described herein are linked via apolypeptide linker having a protease site that is cleavable by aprotease in the blood or target tissue. Such embodiments can be used torelease a therapeutic protein for better delivery or therapeuticproperties or more efficient production.

Additional linkers or spacers, may be introduced at the C-terminus of aFn3 domain between the Fn3 domain and the polypeptide linker. Additionallinkers or spacers may be introduced at the N-terminus of a Fn3 domainbetween the Fn3 domain and the polypeptide linker.

In some embodiments, a therapeutic moiety may be directly or indirectlylinked to a SABA via a polymeric linker. Polymeric linkers can be usedto optimally vary the distance between each component of the fusion tocreate a protein fusion with one or more of the followingcharacteristics: 1) reduced or increased steric hindrance of binding ofone or more protein domains when binding to a protein of interest, 2)increased protein stability or solubility, 3) decreased proteinaggregation, and 4) increased overall avidity or affinity of theprotein.

In some embodiments, a therapeutic moiety is linked to a SABA via abiocompatible polymer such as a polymeric sugar. The polymeric sugar caninclude an enzymatic cleavage site that is cleavable by an enzyme in theblood or target tissue. Such embodiments can be used to release atherapeutic proteins for better delivery or therapeutic properties ormore efficient production.

Summary of Serum Albumin-Binding Adnectins (SABA) Sequences

Many of the SABA sequences referenced in this application are summarizedin Table 3 below. Unless otherwise specified, all N-terminal extensionsare indicated with a single underline, all C-terminal tails/extensionsare indicated with a double underline, and linker sequences are boxed.Loop regions BC, DE and FG are shaded for each core SABA sequence.

TABLE 3 Summary of SABA Exemplary Sequences SEQ ID Sequence NO: NameDescription Sequence 103 SABA1 Core 1 Adnectin

104 SABA1BC Core 1 BC Loop HSYYEQNS 105 SABA1DE Core 1 DE Loop YSQT 106SABA1FG Core 1 FG Loop YGSKYYY 107 SABA2 Core 2 Adnectin

108 SABA2BC Core 2 BC Loop PKYDKTGH 109 SABA2DE Core 2 DE Loop TRQT 110SABA2FG Core 2 FG Loop SKDDYYPHEHR 111 SABA3 Core 3 Adnectin

112 SABA3BC Core 3 BC Loop SNDGPGLS 113 SABA3DE Core 3 DE Loop SSQT 114SABA3FG Core 3 FG Loop SYYTKKAYSAG 115 SABA4 Core 4 Adnectin;contains a scaffold  mutation (bolded); scaffold-perfectversion is SABA5

116 SABA4BC Core 4 BC Loop EDDSYYSR 117 SABA4DE Core 4 DE Loop SDLY 118SABA4FG Core 4 FG Loop YDVTDLIMHE 119 SABA5 Core 5 Adnectin;see description for SAVA4; corrected residue is bolded

120 SABA5BC Core 5 BC Loop EDDSYYSR 121 SABA5DE Core 5 DE Loop SDLY 122SABA5FG Core 5 FG Loop YDVTDLIMHE 123 SABA6 Core 6 Adnectin

124 SABA7 Core 7 Adnectin

125 SABA8 Core 8 Adnectin

126 SABA9 Core 9 Adnectin

127 SABA10 Core 10 Adnectin

128 SABA11 Core 11 Adnectin

129 SABA12 Core 12 Adnectin

130 SABA13 Core 13 Adnectin

131 SABA14 Core 14 Adnectin

132 SABA15 Core 15 Adnectin

133 SABA16 Core 16 Adnectin

134 SABA17 Core 17 Adnectin

135 SABA18 Core 18 Adnectin

136 SABA19 Core 19 Adnectin

137 SABA20 Core 20 Adnectin

138 SABA21 Core 21 Adnectin

139 SABA22 Core 22 Adnectin

140 SABA23 Core 23 Adnectin

141 SABA24 Core 24 Adnectin

142 SABA25 Core 25 Adnectin

143 SABA26 Core 26 Adnectin

Exemplary Adnectin N-Terminal Extension Sequences 144 AdNT1Exemplary leader MGVSDVPRDL 145 AdNT2 Exemplary leader GVSDVPRDL 146AdNT3 Exemplary leader VSDVPRDL 147 AdNT4 Exemplary leader SDVPRDL 148AdNTS Exemplary leader DVPRDL 149 AdNT6 Exemplary leader VPRDL 150 AdNT7Exemplary leader PRDL 151 AdNT8 Exemplary leader RDL 152 AdNT9Exemplary leader DL Exemplary Adnectin C-Terminal Extension Sequences153 AdCT1 Exemplary tail EIDKPSQ 154 AdCT2 Exemplary tail EIDKPS 155AdCT3 Exemplary tail EIDKPC 156 AdCT4 Exemplary tail EIDKP 157 AdCTSExemplary tail EIDK 158 AdCT6 Exemplary tail EI 159 AdCT7 Exemplary tailEIEKPSQ 160 AdCT8 Exemplary tail EIDKPSQLE 161 AdCT9 Exemplary tailEIEDEDEDEDED 162 AdCT10 Exemplary tail EIEKPSQEDEDEDEDED 163 AdCT11Exemplary tail EGSGS 164 L1 G(GS)₂ GGSGS 165 L2 G(GS)₄ GGSGSGSGS 166 L3G(GS)₆ GGSGSGSGSGSGS 167 L4 G(GS)₇ GGSGSGSGSGSGSGS 168 L5 G(GS)₇GGGSGSGSGSGSGSGSG 169 L6 GSGS GSGS 170 L7 (GS)₄ GSGSGSGS 171 L8 (GS)₇GSGSGSGSGSGSGS 172 L9 GS(A)9GS GSAAAAAAAAAGS 173 L10 (GSE)₅GSEGSEGSEGSEGSE 174 L11 (PAS)₅ PASPASPASPASPAS 175 L12 (GSP)₅GSPGSPGSPGSPGSP 176 L13 GS(TVAAPS)₂ GSTVAAPSTVAAPS 177 L14 (GGSE)₂GGSEGGSE 178 L15 (ST)₃G STSTSTG 179 L16 (GGGGS)₇GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGS 180 L17 (GGGGS)₅GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 181 L18 (GGGGS)₃G GGGGSGGGGSGGGGSG 182L19 (GP)₃G GPGPGPG 183 L20 (GP)₅G GPGPGPGPGPG 184 L21 P(ST)₃ PSTSTST 185L22 (PA)₃ PAPAPA 186 L23 (PA)₆ PAPAPAPAPAPA 187 L24 (PA)₉PAPAPAPAPAPAPAPAPA Exemplary Extensions to Adnectin Core Sequences 188SABA1.1 Adnectin core 1 sequence having AdNT1 and AdCT1terminal sequences  with His6 tag MGVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTAT ISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIDKPSQHHHHHH 189 SABA1.2 Adnectin core 1 sequence havingAdNT1 and AdCT8 terminal sequences  MGVSDVPRDLEVVAATPTSLLISWHSYYEQNSYYRITYGETGGNSPVQEFTVPYSQTTAT ISGLKPGVDYTITVYAVYGSKYYYPISINYRTEIEDEDEDEDED 190 SABA1.3 Adnectin core 1 sequence havingAdNT1 and AdCT9 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWHSYYEQ NSYYRITYGETGGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINY RTEIEDEDEDEDEDHHHHHH 191 SABA2.1Adnectin core 2 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWPKYDKTGHYYRITYGETGGNSPVQEFTVPTRQTTAT ISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPSQHHHHHH 192 SABA3.1 Adnectin core 3 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWSNDGPG LSYYRITYGETGGNSPVQEFTVPSSQTTATISGLKPGVDYTITVYAVSYYTKKAYSAGPI SINYRTEIDKPSQHHHHHH 193 SABA4.1Adnectin core 4 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEMVAATPTSLLISWEDDSYYSRYYRITYGETGGNSPVQEFTVPSDLYTAT ISGLKPGVDYTITVYAVTYDVTDLIMHEPISINYRTEIDKPSQHHHHHH 194 SABA5.1 Adnectin core 5 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWEDDSYY SRYYRITYGETGGNSPVQEFTVPSDLYTATISGLKPGVDYTITVYAVTYDVTDLIMHEPI SINYRTEIDKPSQHHHHHH 195 SABA6.1Adnectin core 6 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEWAATPTSLLISWYMDEYDVRYYRITYGETGGNSPVQEFTVPNYYNTAT ISGLKPGVDYTITVYAVTRIKANNYMYGPISINYRTEIDKPSQHHHHHH 196 SABA7.1 Adnectin core 7 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWNHLEHV ARYYRITYGETGGNSPVQEFTVPEYPTTATISGLKPGVDYTITVYAVTITMLKYPTQSPI SINYRTEIDKPSQHHHHHH 197 SABA8.1Adnectin core 8 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWGHYRRSGHYYRITYGETGGNSPVQEFTVDPSSYTAT ISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPSQHHHHHH 198 SABA9.1 Adnectin core 9 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWDASHYE RRYYRITYGETGGNSPVQEFTVPRYHHTATISGLKPGVDYTITVYAVTQAQEHYQPPISI NYRTEIDKPSQHHHHHH 199 SABA10.1Adnectin core 10 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWNSYYHSADYYRITYGETGGNSPVQEFTVPYPPTTAT ISGLKPGVDYTITVYAVYSAKSYYPISINYRTEIDKPSQHHHHHH 200 SABA11.1 Adnectin core 11 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWSKYSKH GHYYRITYGETGGNSPVQEFTVPSGNATATISGLKPGVDYTITVYAVEDTNDYPHTHRPI SINYRTEIDKPSQHHHHHH 201 SABA12.1Adnectin core 12 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWHGEPDQTRYYRITYGETGGNSPVQEFTVPPYRRTAT ISGLKPGVDYTITVYAVTSGYTGHYQPISINYRTEIDKPSQHHHHHH 202 SABA13.1 Adnectin core 13 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWSKYSKH GHYYRITYGETGGNSPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSKDDYYPHEHRPI SINYRTEIDKPSQHHHHHH 203 SABA14.1Adnectin core 14 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWYEPYTPIHYYRITYGETGGNSPVQEFTVPGYYGTAT ISGLKPGVDYTITVYAVYGYYQYTPISINYRTEIDKPSQHHHHHH 204 SABA15.1 Adnectin core 15 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWSKYSKH GHYYRITYGETGGNSPVQEFTVPSGNATATISGLKPGVDYTITVYAVSDDNKYYHQHRPI SINYRTEIDKPSQHHHHHH 205 SABA16.1Adnectin core 16 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWGHYRRSGHYYRITYGETGGNSPVQEFTVDPSSYTAT ISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPSQHHHHHH 206 SABA17.1 Adnectin core 17 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWSKYSKH GHYYRITYGETGGNSPVQEFTVPSGNATATISGLKPGVDYTITVYAVEDTNDYPHTHRPI SINYRTEIDKPSQHHHHHH 207 SABA18.1Adnectin core 18 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWYEPGASVYYYRITYGETGGNSPVQEFTVPSYYHTAT ISGLKPGVDYTITVYAVYGYYEYEPISINYRTEIDKPSQHHHHHH 208 SABA19.1 Adnectin core 19 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWQSYYAH SDYYRITYGETGGNSPVQEFTVPYPPQTATISGLKPGVDYTITVYAVYAGSSYYPISINY RTEIDKPSQHHHHHH 209 SABA20.1Adnectin core 20 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWGHYRRSGHYYRITYGETGGNSPVQEFTVDPSSYTAT ISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPSQHHHHHH 210 SABA21.1 Adnectin core 21 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWPEPGTP VYYYRITYGETGGNSPVQEFTVPAYYGTATISGLKPGVDYTITVYAVYGYYDYSPISINY RTEIDKPSQHHHHHH 211 SABA22.1Adnectin core 22 sequence having AdNT1 and AdCT1 terminal sequences with His6 tag MGVSDVPRDLEVVAATPTSLLISWYRYEKTQHYYRITYGETGGNSPVQEFTVPPESGTAT ISGLKPGVDYTITVYAVYAGYEYPHTHRPISINYRTEIDKPSQHHHHHH 212 SABA23.1 Adnectin core 23 sequence havingAdNT1 and AdCT1 terminal sequences  with His6 tagMGVSDVPRDLEVVAATPTSLLISWVKSEEY YRYYRITYGETGGNSPVQEFTVPYYVHTATISGLKPGVDYTITVYAVTEYYYAGAVVSVP ISINYRTEIDKPSQHHHHHH 213 SABA24.1Adnectin core 24 sequence having AdNT1 and AdCT1 terminal sequenceswith His6 tag MGVSDVPRDLEVVAATPTSLLISWYDPYTYGSYYRITYGETGGNSPVQEFTVGPYTTTAT ISGLKPGVDYTITVYAVSYYYSTQPISINYRTEIDKPSQHHHHHH 214 SABA25.1 Adnectin core 25 sequence havingAdNT1 and AdCT1 terminal sequences with His6 tagMGVSDVPRDLEVVAATPTSLLISWSNDGPG LSYYRITYGETGGNSPVQEFTVPSSQTTATISGLKPGVDYTITVYAVSYYTKKAYSAGPI SINYRTEIDKPSQHHHHHH 215 SABA26.1Adnectin core 26 sequence having AdNT1 and AdCT1 terminal sequenceswith His6 tag MGVSDVPRDLEVVAATPTSLLISWPDPYYKPDYYRITYGETGGNSPVQEFTVPRDYTTAT ISGLKPGVDYTITVYAVYSYYGYYPISINYRTEIDKPSQHHHHHH

EXAMPLES Example 1 Manufacturing Process

Fermentation and Harvest

A production fermentation is prepared with sterile basal medium. A vialis thawed and used to inoculate a transfer vessel containing growthmedium. The inoculum is immediately transferred to the productionfermentation. The culture is maintained at a temperature of 34° C. withagitation and allowed to grow to an OD₆₀₀ of 5-10 (one OD unit isapproximately 1×10⁹ cells/mL) is reached. The addition of feed medium isinitiated at this OD. The fermentation proceeds to OD₆₀₀=25 at whichpoint the culture is induced to produce the Adnectin by the addition ofisopropyl β-D-1-thiogalactopyranoside (IPTG). The temperature of thevessel is increased from 34° C. to 39° C. at the time of induction.Samples are taken aseptically every hour and tested for cell density.

After 9-12 hrs of induced fermentation the vessel is prepared forharvest by reducing the temperature to 25° C., addition ofethylenediaminetetraacetic acid (EDTA) to a final concentration of 10mM, pH increase to 7.8 by the addition of sodium hydroxide and reductionof agitation. After a one hour hold period the fermentor content isdrained into a collection vessel.

Preparation of Inclusion Bodies

Cell disruption of the harvest pool is done by passing the materialthrough a MICROFLUIDIZER® which disrupts the cells and the releasestheir contents. Following cell disruption the inclusion bodies arecollected using a disc stack centrifuge to separate solids and liquidphases in a continuous process by extremely high centrifugal forces.Inclusion bodies are then washed twice with buffer (20-25° C.) and twicewith water (20-25° C.). Each time the washed inclusion bodies arecollected by centrifugation. The washed inclusion bodies are recoveredas a slurry.

Solubilizaton of Inclusion Bodies and Protein Refolding

Solubilization buffer is added to the inclusion body slurry followed bystirring at room temperature for 1 hr. An OD₂₈₀=20 (total protein) istargeted during this process.

The protein refolding is performed using a two step dilution process.Dilution buffer is added to the solubilized inclusion bodies at a ratioof one part solubilized inclusion bodies to one half part dilutionbuffer (v/v). A second dilution is carried out by adding solubilizedinclusion bodies to refold buffer to target an OD₂₈₀=0.7 (totalprotein). The dilutions are carried out while stirring at roomtemperature. Following thorough mixing for one hour, the stirring isstopped and the protein solution is held at room temperature overnight.The solubilized and refolded Adnectin is passed through a 0.8 μm-0.22 μmfilter and tested for protein content by A₂₈₀ and RP-HPLC.

Purification and Conjugation to PEG

Refolded and filtered Adenctin is directly loaded onto a cation exchange(CEX1) column for initial capture. The bound material is washed withwash buffer and eluted with 50 mM sodium acetate, 500 mM sodiumchloride, 1.5% propylene glycol, pH 5.5. The eluate pool is assayed forpurity, identity, concentration, and endotoxin.

The eluate from the capture chromatography is further purified usinghydrophobic interaction chromatography (HIC). The CEX1 eluate isdirectly loaded on the HIC column, washed and subsequently eluted with50 mM sodium acetate, 30% propylene glycol, pH 5.5. The eluate pool isassayed for purity, identity and concentration.

The purified Adenctin is then formatted directly with a maleimidederivative of a 40 kDa branched PEG (mPEG2-MAL). The HIC eluate isstirred at room temperature and the mPEG2-MAL is added. After 1 hr ofmixing at room temperature, the reaction mixture is allowed to incubateovernight at the same temperature. The PEGylation solution is thenprocessed on the final CEX column (CEX2). Samples are taken for proteincontent, purity and endotoxin.

The pH and conductivity of the PEGylation solution are adjusted to 4.0and 1.0 mS/cm respectively, with 75 mM acetic acid prior to loading onthe final cation exchange column (CEX2) for repurification. Once loaded,the bound material is washed with buffer and subsequently eluted with 50mM sodium acetate, 25 mM sodium chloride, pH 5.0. Samples are taken forprotein content, purity and endotoxin.

The CEX2 eluate is concentrated to 15 mg/mL in a tangential flowfiltration unit equipped with a 30 kDa nominal molecular weight cut offmembrane with a V-screen. The bulk drug substance in 50 mM sodiumacetate, 25 mM sodium chloride, pH 5.0. is passed through a 0.22 μmfilter and frozen at −80° C.

Example 2 Gene, Vector and Host Cell

A plasmid encoding the protein under the control of the T7 promoter wasgenerated for use in strain construction. This plasmid DNA was used totransform competent E. coli K-12 MG1655 cells (F-lambda-, ilvG-rfb-50rph-1). The host strain was designed to allow induction of expressionfrom genes upon addition of IPTG. The transformed MG1655 strain isresistant to kanamycin. The protein expression vector is shown in FIG.2. A single colony selection from plates is used to inoculate afermentation culture which is then aliquoted and frozen away to be usedas a research cell bank.

Example 3 Biophysical and Biochemical Characterization

The structure and quality of the protein of the invention were examinedby several comprehensive analytical methods.

MALDI-MS

Mass spectral profiles were analyzed by MALDI. To evaluate precision ofMALDI analysis on the samples, 20 individual spots were placed onto thesteel plate for each sample and analyzed sequentially. A total of 20spectra were generated.

Peptide Mapping

Peptide mapping was used to confirm correct expression of the amino acidsequence (primary structure) predicted from the cDNA sequence for theprotein of the invention as well as the corresponding unPEGylatedprotein. In order to obtain complete sequence coverage, trypsin(cleavage to C-terminal side of Lys and Arg residues) and endoproteinaseGlu-C (cleavage to C-terminal side of Glu residues) were employed toyield two overlapping sets of peptide fragments. Peptide mapping wasalso used to determine covalent post-translational modificationsincluding residual N-terminal methionine, disulfide-bridging,deamidation of asparagine, methionine oxidation (etc.). Peptides wereidentified and characterized by liquid chromatography mass spectrometry(LC-MS) via molecular weight and tandem mass spectrometry (MSMS) whichprovides partial sequence information via collision-induced dissociation(CID).

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) wasused to visualize molecular weight banding patterns of unPEGylated andPEGylated anit-IL-23 Adnectins. The samples were prepared in a samplebuffer with or without a reducing agent. After heating in SDS, thesamples and molecular weight markers were electrophoretically analyzedon pre-cast, gradient (4-20%) polyacrylamide gels. Afterelectrophoresis, the gels were fixed and stained using Coomassie Blue.The equivalence of the banding patterns of samples was assessedvisually.

Size-Exclusion Chromatography/Multi-Angle Light Scattering (SECMALS)

Size-exclusion chromatography (SEC) was used for the quantitativeanalysis of monomer, High Molecular Weight (HMW), and Low MolecularWeight (LMW) species. Following SEC separation, the molecular mass ofseparated species was determined by multi-angle light scattering intandem with a differential refractometer.

Example 4 In Vitro Nonclinical Pharmacology

K_(D) by SPR

The binding characteristics were characterized by Surface PlasmonResonance (SPR). Human IL-23 was immobilized at two to four levels inone dimension of a ProteOn XPR (Bio-Rad) chip surfaces and exposed to 6different concentrations of anti-IL-23 adnectins in the other dimensionof the same SPR chip surface. This allowed kinetic determination in theabsence of regeneration. Duplicate chips were used for kineticdeterminations at 25° C. and 37° C. Evaluation of the kinetic parameterswas performed using the Langmuir interaction model and constantparameter fitting with the ProteOn Manager software.

As shown in Table 4 below, the off-rates for these anti-IL-23 adnectinsare slow (on the order of 10⁻⁵ s⁻¹) at 25° C. Even at 37° C. the offrates were close to the limit of detection for SPR technologies so it ispossible that the reported dissociation constant measurements areunder-estimates.

TABLE 4 Kinetic Parameters of Anti-IL-23 Adnectin Against DirectlyImmobilized Human IL-23 Anti-IL-23 Analysis adnectin temp (° C.) k_(on)(M⁻¹ s⁻¹) k_(off) (s⁻¹) K_(D) (nM) 1490B03 25 2.8 ± 0.6E+04 8.2 ±1.1E−06 0.03 ± 0.002 1571G04 25 5.7 ± 0.6E+04 1.2 ± 0.2E−05 0.2 ± 0.05ATI000934 25 9.4E+03 1.8E−05 1.9 ATI001014 25 9.4 ± 0.2E+03 1.7 ±0.3E−05 1.8 ± 0.2  ATI001047 25  1.3 ± 0.03E+04   2 ± 0.1E−05 1.6 ± 0.1 ATI001045 25 1.5 ± 0.2E+05 2.5 ± 0.4E−05 0.17 ± 0.01  ATI001045 37 2.03± 0.01E+05 5.5 ± 0.6E−05 0.27 ± 0.03 Solution Phase Affinity

The solution affinity of ATI001045 for human IL-23 was measured using aKinetic Exclusion Assay (KinExA). In one format duplicate titrations ofhIL-23 were performed for each of three concentrations. The relativeunbound ATI001045 concentration was measured by capture on a human IL-23solid matrix followed by detection with a fluorescently labeled antibodythat recognizes the Adnectin scaffold. Due to technical limitations, thelowest concentration that could be tested was 0.75 nM. Hence, while theglobal K_(D) analysis shown in Table 5, gives an estimate of 51 pM forthe K_(D), the affinity could be as low as single digit pM or as high as150 pM within a 95% confidence interval.

TABLE 5 Solution Phase Affinity Measurements for ATI001045 K_(D)  51 pM95% confidence interval: K_(D) high 153 pM K_(D) 10W  1 pM

The solution affinity of ATI001045 and ATI001047 for human IL-23 wasalso measured using an alternate format in the KinExA. Duplicatetitrations of adnectins were performed for each of three (ATI001045) orsingle (ATI001047) concentrations of human IL-23 (quadruplicate for thelowest concentration). The relative unbound human IL-23 concentrationwas measured by capture on a non-PEGylated ATI001045 solid matrixfollowed by detection with a fluorescently labeled antibody thatrecognizes the p40 subunit of hIL-23. The global K_(D) analysis shown inTable 6 gives a K_(D) of 9.4 pM with a 95% confidence interval of 22-2.4pM for ATI001045 and a K_(D) of 36.3 pM with a 95% confidence intervalof 60.1 to 19.4 pM.

TABLE 6 Solution Phase Affinity Measurements ATI001045 ATI001047 K_(D)9.4 pM 36.33 pM 95% confidence interval: K_(D) high  22 pM 60.07 pMK_(D) low 2.4 pM 19.44 pMSTAT3 Phosphorylation on Kit225 Cells

Parham et al. (“A receptor for the heterodimeric cytokine IL-23 iscomposed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R”,J. Immunol., 168(11):5699-5708 (Jun. 1, 2002)) cloned the IL-23R fromthe human IL-2 dependent T-cell line, Kit225. These cells have beencharacterized for expression of both IL-12RB1 and IL-23R by FACSanalysis and responded to IL-23 by stimulation of pSTAT3 and to IL-12 bystimulation of pSTAT4. Kit225 cells were seeded into 96 well plates andquiesced in the absence of FBS and IL-2 for 3 hrs at 37° C. Followingthis incubation, human recombinant IL-23 (or IL-23 preincubated withantagonist for 1 hr) was applied and the cells returned to the incubatorfor 15 minutes at 37° C. to stimulate the phosphorylation of STAT3(abbreviated as p-STAT3). Each condition was assayed in duplicate in96-well plates. Stimulation was stopped by placing the cells on ice andaddition of ice-cold PBS. Finally, the cells were pelleted and lysedfollowing standard protocols and pSTAT3 production detected by ELISA.

The optimal concentration of IL-23 for stimulation was 35 pM Inhibitionof the IL-23 induced pSTAT3 was demonstrated by a titration of anti-p40monoclonal antibody (mAb1510) as well as an anti-p19 polyclonal antibody(AF1716). ATI001045, ATI001047, ATI001014 and ATI001016 had equivalentactivity with an IC₅₀ of ˜300 pM, approximately 150 fold more potentthan the anti-p 19 polyclonal antibody while ATI001015 had an IC₅₀ of˜1.2 nM, approximately 40 fold more potent than the anti-p19 polyclonalantibody. Adnectin ATI000934 is ⅓rd the potency of ATI001045, with anIC₅₀ of 1 nM (Table 7).

TABLE 7 Inhibition of IL-23 Induced STAT3 Phosphorylation by Anti-IL-23Antagonists pSTAT3 IC₅₀ ± SD (nM) ATI001045 0.28 ± 0.14 ATI001047 0.36ATI001014 0.3 ± 0.1 ATI001015 1.24 ATI001016 0.3 ± 0.1 ATI000934 0.8 ±0.2 Anti-p40 (mAb1510) 0.19 + 0.04 anti-p19 (AF1716) 52 ± 13STAT3 Phosphorylation on Human PBMCs

A secondary cell-based confirmatory assay was developed with the goal ofevaluating phosphorylation of STAT3 as a mechanism of action in primaryhuman cells. Peripheral blood mononuclear cells (PBMC) from healthydonors consist primarily of naïve and quiescent T-cells that nominallyexpress low levels of IL-23R and do not appreciably respond whenstimulated with exogenous IL-23. However, polyclonal activation of naïvePBMC with IL-2 results in activation and differentiation of naïveT-cells with subsequent increased expression of IL-23R. These activatedcells are then susceptible to stimulation with exogenous IL-23 whichactivates the STAT pathway, resulting in phosphorylation of STAT3.

Commercially available antibodies (AF1716, an anti-p19 pAb and mAb1510,an anti-p40 mAb, both from R&D Systems) were used as positive controlsfor inhibition of IL-23 induced STAT3 phosphorylation. The inhibitoryactivity of six adnectins was compared in ten separate experiments usingblood from multiple donors (summarized in Table 8). Exemplary data for asubset are shown in FIG. 4. The anti-IL-23 adnectins were significantly(>150-fold) more potent than the anti-p19 in inhibiting STAT3phosphorylation but similar to 5 fold less potent than the anti-p40monoclonal antibody.

TABLE 8 Inhibition of PBMC pSTAT3 by Anti-IL-23 Antagonists pSTAT3 IC₅₀± SD (nM) 1490B03 0.03 ± 0.02 1571G04 0.09 ± 0.06 1572G06  0.07 ± .0.02ATI 934   0.14 ± .0.12 ATI 1016 0.06 ± 0.03 ATI 1045 0.07 ± 0.03 MAB15100.03 ± 0.04 AF1716 21.4 ± 9.4 IL-23 Induced Cytokine Production by Mouse Splenocytes

Initial cellular assays with primary cells were designed to evaluate thecapacity of anti-IL-23 adnectins to inhibit IL-23-dependent cytokinesecretion from murine Th17 cells. To differentiate murine Th17 cells foranalysis, CD4+ T cells were enriched with magnetic beads, co-culturedwith irradiated splenocytes, and activated with anti-CD3 in presence ofTGF-β and IL-6 and neutralizing antibodies for IL-4 and IFN-γ. After 6days in culture, the polarized Th17 cells were harvested, re-seeded in a96-well plate and stimulated with 100 ng/ml human IL-23 and 5 ng/mlmurine IL-2. The addition of IL-2 was required to maintain cellviability and enable robust cytokine production in response to IL-23 butdid not strongly induce IL-17A or IL-22 production alone. Because IL-2induces a low level of cytokine secretion, each sample set includedcells stimulated with IL-2 alone to control for baseline levels ofcytokine produced in the absence of IL-23. The IL-23-dependent responsewas evaluated by calculating the difference between the level ofcytokine induced by the combination of IL-2 and IL-23 and the baselinelevel induced by IL-2 alone. A dose range of adnectins were added duringre-stimulation of the Th17 cells with IL-2 and IL-23 to test theirinhibitory potential. A dose range of human anti-p40 antibody (R&DSystems MAB1510) was run in parallel as positive controls for assessingIL-23 inhibition. Each condition was tested in triplicate wells of a96-well plate. After 4 days, the conditioned media from the triplicateswas pooled, cleared of cellular debris, and assayed for both IL-17A andIL-22 concentrations by ELISA.

Stimulation of Th17 cells with IL-2 and IL-23 induced a 2- to 3-foldincrease of IL-17A and at least a 5-fold enhancement of IL-22 comparedto the levels induced by IL-2 alone. ATI000934, ATI001014, ATI001015,ATI001016, ATI001045 and the positive control anti-p40 monoclonalantibody mediated dose-dependent decreases in IL-23-dependent IL-17A andIL-22 secretion. IC₅₀ values for inhibition of both IL-17A and IL-22secretion were calculated for each adnectin as well as the anti-p40control and these data were summarized in Table 9. All of the adnectinstested were within 2-fold as potent as the anti-p40 control forinhibition of IL-23-dependent IL-17A secretion and within 2-to 3-fold aspotent for inhibition of IL-23-dependent IL-22 production.

TABLE 9 Inhibition of IL-23-Dependent Cytokines by Anti-IL-23 AdnectinsIC₅₀ ± S.D. IC₅₀ ± S.D. Adnectin/Ab IL-17 (nM) IL-22 (nM) anti-p40(MAB1510) 2.3 ± 0.7 (n = 5) 1.9 ± 0.7 (n = 5) ATI000934 5.3 ± 1.6 (n =2) N.D. ATI001045 1.3 ± 0.3 (n = 3) 2.2 ± 1.1 (n = 3) ATI001014 3.7 ±0.0 (n = 2) 6.5 ± 2.2 (n = 4) ATI001015 2.0 ± 0.1 (n = 2) 5.5 ± 2.4 (n =4) ATI001016 2.0 ± 2.0 (n = 4) 3.0 ± 1.8 (n = 5)IL-23 Induced Cytokine Production by Human T Cells

PBMCs were obtained by density-gradient separation of EDTA-treated wholeblood from normal healthy donors. T cells were prepared from E+fractions of PBMC rosetted with sheep red blood cells (SRBC). The Tcells were plated at 100,000 cells per well into 96-well flat bottomplates that were coated with anti-CD3 (OKT at 10 μg/ml) for 1 hour at37° C. and washed with PBS. Mixtures of RPMI-FCS media containinganti-CD28 (9.3 at 1 μg/ml) and IL-1β (10 ng/ml) or IL-1β+IL-23 (1 ng/ml)were prepared. This combination of cytokines has been shown to promotethe differentiation of human T cells into IL-17-secreting T cells.ATI001045, starting concentration of 1 μg/ml was added to the mixturecontaining IL-1β+IL-23. IL-17 was detected in supernatants using DUOSET®ELISA development kits (R&D Systems). ATI001045 inhibited IL-17production with an EC₅₀ of 2.0±1.6 nM (n=4 different donors), using theIL-1β alone as background. The commercial anti-p40 antibody (MAB1510)was used as an internal control and inhibited IL-17 production with anEC50 of 2.2±1.4 nM (n=3). Exemplary data from donor 228 is shown in FIG.6.

Selectivity of Anti-IL-23 Adnectin for IL-23 Over IL-12

Adnectins listed in Table 2 as well as ATI001016 were used to examinethe biochemical selectivity towards IL-23/IL-12. The binding analysisinvolved the capture of anti-IL-23 Adnectins on immobilized anti-Hisantibody followed by flow of IL-23 or IL-12 over the Adnectin. Theselectivity of the Adnectins for IL-23 was assessed by comparing thebinding signal for a 100 fold higher concentration of IL-12 over IL-23.Exemplary data in FIG. 7 shows that ATI001016 displayed robust binding(˜40 RU) towards 10 nM human IL-23 while no detectable binding wasobserved for 1 μM human IL-12.

NK-92 cells are a human natural killer cell line known to respond toIL-12 in an IL-2 dependent fashion by secreting IFN-γ. Cells aretypically washed to remove IL-2 then seeded into 96-well plates, thentreated with 25 pM recombinant human IL-12 (or IL-12 preincubated withantagonists) and incubated for an additional 20 hours. Clarifiedsupernatants are assayed for IFN-γ by ELISA.

A 4 point, 5 fold dilution series starting at 5 uM was prepared of eachof the adnectin clones listed in Table 2 and incubated with 25 pM IL-12for 30 minutes at 37° C. prior to the addition to NK-92 cells. A 12point, 5 fold dilution series starting at 5 μM of ATI001045 andATI001016 were incubated with 25 pM IL-12 for 30 minutes at 37° C. priorto the addition to NK-92 cells. None of the clones listed in Table 2 norATI001045 or ATI001016 detectably inhibited IFN-γ secretion at any ofthe concentrations tested demonstrating that these anti-IL-23 adnectinsdo not inhibit the interaction of IL-12 with the receptors on thesurface of NK-92 cells. They appear equivalent to a negative control and100 nM anti-p19 polyclonal antibody. As a positive control, anti-p40monoclonal antibody (mAb1510) inhibited IL-12 induced IFN-γ secretionwith an IC₅₀ of 0.07 nM (FIG. 8).

Anti-IL-23 Adnectin Block IL-23 Induced IL-17 in a Pharmacodynamic Model

Female C57B1/6 mice were injected intraperitoneally (IP) withrecombinant murine IL-2 and human IL-23 according with the followingschedule.

TABLE 10 Dosing and Injection Schedule Time = −24 h Time = 0 h Time = 7h Time = 23 h Murine IL-2 5 μg  5 μg 10 μg  5 μg Human IL-23 0 10 μg 10μg 10 μg

All mice were euthanized 7-8 hours following the final dose of IL-2 andIL-23 at Time=30 h. Serum was collected and assayed for IL-17 and IL-23by ELISA.

Human IL-23 binds to the mouse receptor and induces the production ofcytokines such as IL-17 and IL-22. Splenocytes from animals dosedintraperitoneally (IP) with IL-2 and human IL-23 secrete IL-17 whenstimulated in culture ex vivo with anti-mouse CD3e. Significant levelsof IL-17 can be detected in the serum of animals that were subjected tothe treatment regimen described in Table 10 in which C57B1/6 mice areprimed with IL-2 24 hours prior to 3 dual injections of IL-2+IL-23 overan additional span of 24 hours. Presumably, IL-2 polyclonally activatesand expands Th populations in situ and up-regulates the expression ofIL-23 receptor. This provides a method where the mechanisms of drugaction and the relationship between drug concentration and effect in anin vivo setting can be investigated. The model was validated with ananti-p40 monoclonal antibody, mAb1510 (data not shown). In eightseparate experiments, five anti-IL-23 adnectins were tested for theirability to inhibit the production of murine IL-17 when dosed SC at 0.5,0.15, 0.05 and 0.015 mg/kg 2 hours prior to the initial dose ofIL-2+IL-23 in eight separate experiments. Exemplary dose response datafor ATI001045 is shown in FIG. 9 a (calculated average ED50 of 0.03mg/kg). All anti-IL-23 adnectins tested showed dose dependent inhibitionof human IL-23 murine IL-17 production in serum though the extent ofinhibition was variable across adnectins.

Activity of Anti-IL-23 Adnectin in the IL-23 Induced Skin AcanthosisModel

The intra-dermal injection of IL-23 into the skin of the back or intothe external ear pinna of mice induces dermal inflammation andhyperplasia of the epidermis (acanthosis) (Zheng, Y., “Interleukin-22, aTH17 cytokine, mediates IL-23-induced dermal inflammation andacanthosis”, Nature, Vol. 445/8 (February 2007)). In these studies,recombinant human IL-23 (rHuIL-23) was injected into mouse ears toexplore the downstream consequences of aberrant cutaneous IL-23exposure.

Six to eight week old C57BL/6 female mice were injected with 5 ug ofdual chain, recombinant, human IL-23 into the right ear every other dayuntil Day 12. PBS was injected into the contra-lateral ear as a control.In one study, treatment with ATI001045 began approximately 2 hoursbefore the first IL-23 injection and continued 3 times per week untilDay 12. ATI001045 was administered SC at doses of 0.1, 0.3, 1, 3 mg/kg.In a second study vehicle or ATI000934-123 (1753E02) was administered IPat 1, 3, or 10 mg/kg approximately 1 hour prior to IL-23 administrationand 3 times per week thereafter until Day 10. Anti-HuIL-12/IL-23 p40Antibody (R&D mAb1510) at 10 mg/kg was given IP on Day 0 and 4 as apositive control. Ear thickness (in thousandths of an inch) was measuredevery-other-day, prior to the next ear injection, using a MITUTOYO®(#2412F) dial caliper. Ear thickness was calculated by subtracting thevalue of the control ear from the measurement for the IL-23 injected earfor each animal At the end of the study (Day 14 for ATI001045 and Day 12for ATI000934), following euthanasia with CO₂ gas, ears were excised atthe hairline and formalin fixed/paraffin-embedded tissues were examinedhistologically on H&E stained slides.

Overall, doses of 1, 3, and 10 mg/kg of ATI000934 provided a similarlevel of inhibition of IL-23-induced ear thickening in this study (FIG.10). Ear thickness in all treatment groups was significantly (p<0.01ANOVA/Dunnett's) less than Vehicle, including the anti-p40 group, fromDay 5 through the end of the study on Day 12. On Day 12, terminal plasmasamples were obtained 48 hours post last dose and analyzed forcirculating levels of ATI000934 which were determined to be 11, 18, 36ug/ml respectively.

Following the last measurement on Day 12, ears were collected atnecropsy for routine histologic examination from 10 animals per group.The majority of animals administered ATI000934 had acanthosis and dermalinfiltrates, but the histologic severity score was reduced from thatobserved in vehicle treated animals. There was no apparent doseresponse. All of the animals administered anti-p40 also had acanthosisand dermal infiltrates, but the histologic severity score was alsoreduced from that observed in vehicle treated animals.

ATI001045 (1 mg/kg and 3 mg/kg) dose-dependently reduced ear thicknesscompared to Vehicle (PBS) treated animals from Day 5 through Day 14(p<0.01 vs. Vehicle ANOVA/Dunnett's, FIG. 10). In contrast, the 0.1mg/kg dose level was not statistically different (p>0.05) from Vehicletreatment on any study day. Treatment with 0.3 mg/kg providedintermediate reduction that was statistically less than Vehicle on Days,5, 7, 9. Serum samples collected 48 hours post last dose were evaluatedfor circulating levels of ATI001045 which were determined to be 0.698,2.72, 8, 22.5 ug/ml for doses of 0.1, 0.3, 1, 3 mg/kg respectively.Histological analysis revealed that administration of ATI001045 resultedin a dose dependent reduction of IL-23 induced cellular infiltrates andacanthosis which correlated with the ear thickness score.

Example 5 Material and Methods Used Herein

High Throughput Protein Production (HTPP)

Selected binders were cloned into pET9d vector and transformed into E.coli BL21 DE3 plysS cells were inoculated in 5 ml LB medium containing50 μg/mL kanamycin in a 24-well format and grown at 37° C. overnight.Fresh 5 ml LB medium (50 μg/mL kanamycin) cultures were prepared forinducible expression by aspiration 200 μl from the overnight culture anddispensing it into the appropriate well. The cultures were grown at 37°C. until A600 0.6-0.9. After induction with 1 mMisopropyl-β-thiogalactoside (IPTG) the culture was expressed for 6 hoursat 30° C. and harvested by centrifugation for 10 minutes at 2750 g at 4°C.

Cell pellets (in 24-well format) were lysed by resusupension in 450 μlof Lysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete ProteaseInhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mMImidazole, 1 mg/ml lysozyme, 30 μg/ml DNAse, 2 μg/ml aprotonin, pH 8.0)and shaken at room temperature for 1-3 hours. Lysates were clarified andre-racked into a 96-well format by transfer into a 96-well Whatman GF/DUNIFILTERO fitted with a 96-well, 1.2 ml catch plate and filtered bypositive pressure. The clarified lysates were transferred to a 96-wellNi-Chelating Plate that had been equilibrated with equilibration buffer(50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM Imidazole, pH 8.0) and was incubatedfor 5 min. Unbound material was removed by positive pressure. The resinwas washed 2×0.3 ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl,5 mM CHAPS, 40 mM Imidazole, pH 8.0) with each wash removed by positivepressure. Prior to elution each well was washed with 50n1 Elution buffer(PBS+20 mM EDTA), incubated for 5 min and this wash was discarded bypositive pressure. Protein was eluted by applying an additional 100n1 ofElution buffer to each well. After a 30 minute incubation at roomtemperature the plate(s) were centrifuged for 5 minutes at 200 g andeluted protein is collected in 96-well catch plates containing 5 μl of0.5M MgCl2 added to the bottom of elution catch plate prior to elution.Eluted protein was quantified using a total protein assay (BCA) with SGEas the protein standard.

Midscale Expression and Purification of Insoluble Fibronectin-BasedScaffold Protein Binders

For expression, selected clone(s), followed by the HIS6tag, were clonedinto a pET9d vector and were expressed in E. coli BL21 DE3 plysS cells.Twenty ml of an inoculum culture (generated from a single plated colony)was used to inoculate 1 liter of LB medium or TB-Overnight ExpressionMedia (auto induction) containing 50 μg/ml Kanamycin and 34 μg/mlchloramphenicol. Cultures in LB medium were incubated at 37° C. untilA600 0.6-1.0 at which time they then induced with 1 mMisopropyl-β-thiogalactoside (IPTG) and grown for 4 hours at 30° C.Cultures grown in TB-Overnight Expression Media were incubated at 37° C.for 5 hours at which time the temperature was lowered to 18° C. grownfir 19 hours. Cultures were harvested by centrifugation for 30 minutesat 10,000 g at 4° C. Cell pellets were frozen at −80° C. the cell pelletwas resuspended in 25 ml of lysis buffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1×Complete Protease Inhibitor Cocktail-EDTA free (Roche), pH 7.4) using anULTRA-TURRAX® homogenizer (IKA works) on ice. Cell lysis was achieved byhigh pressure homongenization (≧18,000 psi) using a Model M-110SMICROFLUIDIZER® (Microfluidics). The insoluble fraction was separated bycentrifugation for 30 minutes at ≧23,300 g at 4° C. The insoluble pelletrecovered from centrifugation of the lysate was washed with 20 mM sodiumphosphate/500 mM NaCl, pH7.4. The pellet was resolubilized in 6.0Mguanidine hydrochloride in 20 mM sodium phosphate/500 mM NaCl pH 7.4with sonication followed by incubation at 37 degrees for 1-2 hours. Theresolubilized pellet was filtered to 0.45 μm and loaded onto a HISTRAP®column equilibrated with the 20 mM sodium phosphate/500 mM NaCl/6.0Mguanidine pH7.4 buffer. After loading, the column was washed for anadditional 25 CV with the same buffer. Bound protein was eluted with 50mM Imidazole in 20 mM sodium phosphate/500 mM NaCl/6.0M guan-HCl pH7.4.The purified protein was refolded by dialysis against 50 mM sodiumacetate/150 mM NaCl pH 4.5 or PBS pH 7.2.

Midscale Expression and Purification of Soluble Fibronectin-BaseScaffold Protein Binders

As an alternative to purification of insoluble binders, the purificationof soluble binders may also be used. For expression, selected clone(s),followed by the HIS6tag, were cloned into a pET9d vector and wereexpressed in E. coli BL21 DE3 plysS cells. Twenty ml of an inoculumculture (generated from a single plated colony) was used to inoculate 1liter of LB medium or TB-Overnight Expression Media (auto induction)containing 50 μg/ml Kanamycin and 34 μg/ml chloramphenicol. Cultures inLB medium were incubated at 37° C. until A600 0.6-1.0 at which time theywere then induced with 1 mM isopropyl-β-thiogalactoside (IPTG) and grownfor 4 hours at 30° C. Cultures grown in TB-Overnight Expression Mediawere incubated at 37° C. for 5 hours at which time the temperature waslowered to 18° C. grown fir 19 hours. Cultures were harvested bycentrifugation for 30 minutes at 10,000 g at 4° C. Cell pellets arefrozen at −80° C. The cell pellet is resuspended in 25 ml of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), pH 7.4) using an ULTRA-TURRAX® homogenizer(IKA works) on ice. Cell lysis is achieved by high pressurehomongenization (≧18,000 psi) using a Model M-110S MICROFLUIDIZER®(Microfluidics). The soluble fraction is separated by centrifugation for30 minutes at ≧23,300 g at 4° C. The supernatant is clarified via 0.45μm filter. The clarified lysate is loaded onto a HISTRAP® column (GE)pre-equilibrated with the 20 mM sodium phosphate/500 mM NaCl pH 7.4. Thecolumn is then washed with 25 column volumes of the same buffer,followed by 20 column volumes of 20 mM sodium phosphate/500 mM NaCl/25mM Imidazole, pH 7.4 and then 35 column volumes of 20 mM sodiumphosphate/500 mM NaCl/40 mM Imidazole, pH 7.4. Protein is eluted with 15column volumes of 20 mM sodium phosphate/500 mM NaCl/500 mM Imidazole,pH 7.4, fractions are pooled based on absorbance at A280 and aredialyzed against 1×PBS, 50 mM Tris, 150 mM NaCl. pH 8.5 or 50 mM NaOAc;150 mM NaCl; pH4.5. Any precipitate is removed by filtering at 0.22 nm.

Fibronectin-based scaffold proteins (Adnectins) can be pegylated withvarious sizes and types of PEG. To allow for pegylation, the naturallyoccurring residues EIDKPSQ, found at the C-terminus end of 10FN3proteins can be modified by a single point mutation of an amino acid,typically a serine, to a cysteine. PEGylation of the protein at thesingle cysteine residue is accomplished by conjugating variousmaleimide-derivatized PEG forms, combining the PEG reagent with theprotein solution and incubating. An alternative method is to replace theEIDKPSQ tail with a GSGC linker, and similarly use the cysteine residuefor PEGylation. Adnectins containing an engineered cysteine residue wereconjugated with PEG via Michael-addition chemistry between the thiolgroup on the cysteine and the maleimide functional group of the PEGreagent. Briefly, 40 kDa PEG is added in a molar excess to proteinsolution under slightly acidic to neutral conditions. The reaction isallowed to proceed at room temperature for 2 hours to overnight. Thereaction is then applied to an ion exchange column to separate thePEGylated Adnectin from the unreacted PEG-maleimide and non-PEGylatedAdnectin. SE/HPLC methods may also be used. The purified PEGylatedAdnectin is typically analyzed by SDS-PAGE and size exclusionchromatography.

Example 6 Screening and Selection of Candidate Serum Albumin-BindingAdnectin (SABA)

A selection technique known as PROfusion (see, e.g., Roberts et al.,Proc. Natl. Acad. Sci. USA, 94(23):12297-12302 (1997) and WO2008/066752) was applied to a DNA library with variable regions designedinto the BC, DE and FG loops of ¹⁰Fn3. A random library of greater than10¹³ molecules was created from this design, and selection pressure wasapplied against a biotinylated form of HSA to isolate candidate serumalbumin-binding Adnectin (SABA) with desirable binding properties.

High Throughput Protein Production (HTTP) Process

The various HSA binding Adnectins were purified using a high throughputprotein production process (HTPP). Selected binders were cloned intopET9d vector containing a HIS6 tag and transformed into E. coliBL21(DE3)pLysS cells. Transformed cells were inoculated in 5 ml LBmedium containing 50 μg/mL Kanamycin in a 24-well format and grown at37° C. overnight. Fresh 5 ml LB medium (50 μg/mL Kanamycin) cultureswere prepared for inducible expression by aspirating 200 μl from theovernight culture and dispensing it into the appropriate well. Thecultures were grown at 37° C. until A₆₀₀ 0.6-0.9. After induction with 1mM isopropyl-β-thiogalactoside (IPTG), the culture was grown for another4 hours at 30° C. and harvested by centrifugation for 10 minutes at3220×g at 4° C. Cell Pellets were frozen at −80° C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1mg/ml lysozyme, 30 ug/ml DNAse, 2 ug/ml aprotonin, pH 8.0) and shaken atroom temperature for 1 hour. Lysates were clarified and re-racked into a96-well format by transfer into a 96-well Whatman GF/D UNIFILTERO fittedwith a 96-well, 650 μl catch plate and centrifuged for 5 minutes at200×g. The clarified lysates were transferred to a 96-well Ni-ChelatingPlate that had been equilibrated with equilibration buffer (50 mMNaH₂PO₄, 0.5 M NaCl, 10 mM CHAPS, 40 mM Imidazole, pH 8.0) and incubatedfor 5 min. Unbound material was removed. The resin was washed 2×0.3ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5 mM CHAPS, 40mM Imidazole, pH 8.0). Next the resin was washed with 3×0.3 ml/well withPBS. Prior to elution each well was washed with 50 μl Elution buffer(PBS+20 mM EDTA), incubated for 5 mM and this wash discarded by vacuum.Protein was eluted by applying an additional 100 ul of Elution buffer toeach well. After 30 minute incubation at room temperature the plate(s)were centrifuged for 5 minutes at 200×g and eluted protein collected in96-well catch plates containing 5 μl of 0.5M MgCl₂ affixed to the bottomof the Ni-plates. Eluted protein was quantified using a BCA Proteinassay with SGE (control Adnectin) as the protein standard. The SGEAdnectin is a wild-type ¹⁰Fn3 domain (SEQ ID NO: 1) in which integrinbinding domain (amino acids RGD at positions 78-80) have been replacedwith SGE.

HSA, RhSA and MuSA Direct Binding ELISA

For assaying direct binders to HSA, MaxiSorp plates (Nunc International,Rochester, N.Y.) were coated with 10 ug/mL HSA (Sigma, St. Louis, Mo.)in PBS at 4° C. overnight followed by blocking in casein block buffer(Thermo Scientific, Rockford, Ill.) for 1-3 hours at room temperature.For single-point screening assays, purified HTPP Adnectin were diluted1:20 in casein block buffer and allowed to bind to HSA in each well for1 hour at room temperature. For dose response assays, concentrationsranging from 0.1 nM up to 1 μM were used. After washing in PBST toremove unbound Adnectins, anti-His mAb-HRP conjugate (R&D Systems, MN)diluted 1:2500 in casein block buffer was added to the bound His-taggedAdnectin for 1 hour at room temperature. Excess conjugate was removed bywashing with PBST and bound Adnectins detected using TMB detectionreagents (BD Biosciences) according to the manufacturer's instructions.

Identification of Candidate Serum Albumin-Binding Adnectin (SABA)

As a result of the screening for HSA/RhSA/MuSA binding and biophysicalcriteria, four unique serum albumin-binding Adnectins (SABA) wereidentified and chosen to have their half-lives evaluated in mice. Inorder to carry out in vitro and in vivo characterization, midscales wereundertaken for the four SABAs. Table 3 provides the sequences oftwenty-six unique SABA core sequences identified from PROfusion,designated as SABA 1-26. SABA4 had a scaffold mutation that was fixedprior to midscaling. The scaffold-perfect version of SABA4 is SABAS.SABA4 and SABAS have identical sequences in the BC, DE, and FG loops.

Example 7 Production and Formulation of Candidate SABAs

Midscale Protein Production of SABAs

The selected SABAs followed by the His₆tag, were cloned into a pET 9dvector and expressed in E. coli BL21(DE3)pLysS cells (see Table 3 foreach His-tagged SABA sequence designated SABA1.1, SABA2.1, SABA3.1, andSABA5.1). 20 ml of an inoculum culture (generated from a single platedcolony) was used to inoculate 1 liter of LB medium containing 50 μg/mLKanamycin. The culture was grown at 37° C. until A₆₀₀ 0.6-1.0. Afterinduction with 1 mM isopropyl-β-thiogalactoside (IPTG) the culture wasgrown for another 4 hours at 30° C. and harvested by centrifugation for30 minutes at ≧10,000×g at 4° C. Cell Pellets were frozen at −80° C. Thecell pellet was resuspended in 25 mL of lysis buffer (20 mM NaH₂PO₄, 0.5M NaCl, 1× Complete Protease Inhibitor Cocktail-EDTA free (Roche), pH7.4) using an ULTRA-TURRAX® homogenizer (IKA works) on ice. Cell lysiswas achieved by high pressure homogenization (≧18,000 psi) using a ModelM-110S MICROFLUIDIZER® (Microfluidics). The soluble fraction wasseparated by centrifugation for 30 minutes at 23,300×g at 4° C. Thesupernatant was clarified via 0.45 μm filter. The clarified lysate wasloaded onto a HISTRAP® column (GE) pre-equilibrated with 20 mM NaH₂PO₄,0.5 M NaCl, pH 7.4. The column was then washed with 25 column volumes of20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4, followed by 20 column volumes of 20mM NaH₂PO₄, 0.5 M NaCl, 25 mM imidazole pH 7.4, and then 35 columnvolumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole pH 7.4. Proteinwas eluted with 15 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 500 mMimidazole pH 7.4, fractions pooled based on absorbance at A₂₈₀ anddialyzed against 1×PBS, 50 mM Tris, 150 mM NaCl pH 8.5 or 50 mM NaOAc;150 mM NaCl; pH 4.5. Any precipitate was removed by filtering at 0.22μm.

Midscale expression and purification yielded highly pure and activeAdnectins that were expressed in a soluble form and purified from thesoluble fraction of the bacterial cytosol. SEC analysis on a SUPERDEX®200 or SUPERDEX® 75 10/30GL in a mobile phase of 100 mM NaPO₄, 100 mMNaSO₄, 150 mM NaCl, pH 6.8 (GE Healthcare) demonstrated predominantlymonomeric Adnectins.

Formulation of SABA1.2

One specific SABA, SABA1.2 (SEQ ID NO: 180), was chosen for apreliminary formulation screen. SABA1.2 comprises an (ED)₅ extension onthe “core 1” sequence of ¹⁰Fn3. For SABA1.2, a stable formulation of 10mM succinic acid, 8% sorbitol, 5% glycine at pH 6.0 and at a productconcentration of 5 mg/mL was identified. In this formulation the proteinmelting temperature was 75° C. as determined by Differential Scanningcalorimetry (DSC) using a protein concentration of 1.25 mg/mL. Theformulation provided satisfactory physical and chemical stability at 4°C. and 25° C., with an initial aggregate level at 1.2%. After one monthof stability, the level of aggregation was very low (1.6% at 4° C. and3.8% at 25° C.). The protein was also stable in this formulation afterfive cycles of freeze-thaw as transitioned from −80° C. and −20° C. toambient temperature. In addition, in this formulation SABA1.2 wassoluble to at least 20 mg/mL protein concentration at 4° C. and ambienttemperature with no precipitation or increase in aggregation.

Example 8 Biophysical Characterization of Candidate SABAs

Size Exclusion Chromatography

Standard size exclusion chromatography (SEC) was performed on thecandidate SABAs resulting from the midscale process. SEC of midscaledmaterial was performed using a SUPERDEX® 200 10/30 or on a SUPERDEX® 7510/30 column (GE Healthcare) on an Agilent 1100 or 1200 HPLC system withUV detection at A₂₁₄ nm and A₂₈₀ nm and with fluorescence detection(excitation=280 nm, emission=350 nm). A buffer of 100 mM sodium sulfate,100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8 at appropriateflow rate of the SEC column employed. Gel filtration standards (Bio-RadLaboratories, Hercules, Calif.) were used for molecular weightcalibration.

The results of the SEC on the midscaled purified SABAs showedpredominantly monomeric Adnectin and elution in the approximate range of10 kDa vs. globular Gel Filtration standards (BioRad) as showed.

Thermostability

Differential Scanning calorimetry (DSC) analyses of the midscaled SABAswere performed to determine their respective T_(m)'s. A 1 mg/ml solutionwas scanned in a N-DSC II calorimeter (calorimetry Sciences Corp) byramping the temperature from 5° C. to 95° C. at a rate of 1 degree perminute under 3 atm pressure. The data was analyzed vs. a control run ofthe appropriate buffer using a best fit using Orgin Software (OrginLabCorp). The results of the SEC and DSC analyses are summarized in Table11.

TABLE 11 Summary of SEC and DSC Analyses on Candidate SABAs SEC CloneMonomer (%) Dimer (%) DSC (Tm) SABA1.1 92.3 7.7 63.9° C. SABA5.1 88 1270.1° C. SABA2.1 91 9 58.5° C./78.2° C. SABA3.1 99 BLD 65.2° C.

Example 9 Characterization of Candidate SABA1 Binding to Serum Albumin

The kinetics of selected SABA clones purified from HTPP and/or midscaledmaterial were determined by capturing the respective serum albumin(HSA/RhSA/MuSA) on the surface of a Biasensor CM5 chip and flowing aconcentration series of SABAs over both the reference flow cell and thecaptured albumins. In addition, binding to albumin was carried out undervarious pH conditions ranging from pH 5.5 to pH 7.4. HSA-bindingAdnectins SABA2.1, SABA3.1, SABA4.1 (SABA5.1) & SABA1.1 cross reactedwith RhSA but did not cross react with MuSA. SABA2 and SABA4 binding ispH sensitive whereas clone SABA3 demonstrated pH resistance binding toHSA down to pH 6.0. SABA1.1 fits biochemical criteria for pH resistanceand affinity/kinetics down to pH 5.5.

Domain mapping was determined by Biacore. Selected SABA clones purifiedfrom HTPP and/or midscaled material were determined by capturing HSA ora construct consisting of just HSA-domain I & II or HSA-domain III onthe surface of a Biasensor CMS chip and flowing a concentration seriesof the SABAs over both the reference flow cell and the capturedalbumins. Clones SABA2 & SABA1 bound to HSA and the HSA-domain I-IIconstruct but not the HSA-domain III construct. Clones SABA3 & SABA4bound to HSA but not to either the HSA-domain I-II or HSA-domain IIIconstructs. The results are summarized in Table 12.

TABLE 12 Binding Affinity and Kinetics of Candidate SABAs (SABA1.1, 2.1,3.1 and 4.1) Resistant to pH Adnectin Target K_(D) (nM) K_(off) (s⁻¹)7.4→5.5 Epitope on HSA SABA2 HSA 33.8 +/− 20.5 (n = 6) 1.71E−04 −−Domain I-II RhSA 63.6 4.42E−04 SABA3 HSA 863 6.82E−02 +++ Neither domainRhSA 431 3.37E−02 (down to pH 6.0) I-II nor III (interfacial?) SABA4 HSA412 +/− 8 (n = 4)  7.82E−04 −− Neither domain RhSA >1000 3.83E−03 I-IInor III (interfacial?) SABA1 HSA 47.2 +/− 18.2 (n = 9) 4.57E−04 +++Domain I-II RhSA 778 +/− 313 (n = 4) 5.45E−03

Example 10 Examination of the In Vivo t_(1/2) of Candidate SABAs

The half-life of HSA in mice was determined to allow for evaluation ofHSA-binding Adnectins in mice as the HSA-binding Adnectins do not crossreact with MuSA. HSA was injected into the tail vein of approximately 6week old Ncr nude female mice at a 20 mg/kg (FIG. 11A) and 50 mg/kg dose(FIG. 11B), and the concentration of HSA in blood samples taken atintervals post-injection was determined by ELISA. The t_(1/2) of HSAinjected into mice at 20 mg/kg and 50 mg/kg were determined to be ˜24hrs and ˜20 hrs, respectively.

Half-Life Determination of SABA1-4 in Mice

One liter E. coli growth of HSA binding clones SABA1.1, SABA2.1,SABA3.1, and SABA4.1 were prepared, purified and endotoxin removed. EachSABA variant was injected into the tail vein of mice, and theconcentration in blood samples taken at intervals post-injection wasdetermined by ELISA.

The pharmacokinetic profiles of each SABA were compared in the presenceor absence of HSA in approximately 6 week old Ncr nude female mice. Themice that were co-injected with HSA had the HSA premixed with each SABA(HSA in a 3-4 molar excess) because the binding clone was selective forHSA and RhSA and did not bind the mouse serum albumin. The half-life ofSABA1.1 in mice plasma was 0.56 hours whereas the half-life of SABA1.1co-injected with HSA was 5.6 hours, a ˜10-fold increase in half life(FIG. 12A). The half-life of SABA2.1 in mice plasma was 0.24 hourswhereas the half-life of SABA2.1 co-injected with HSA was 2.8 hours, a˜12-fold increase in half life (FIG. 12B). The half-life of SABA3.1 inmice plasma was 0.28 hours whereas the half-life of SABA3.1 co-injectedwith HSA was 0.53 hours, a ˜2-fold increase in half life (FIG. 12C). Thehalf-life of SABA4.1 in mice plasma was 0.66 hours whereas the half-lifeof SABA4 co-injected with HSA was 4.6 hours, a ˜7-fold increase in halflife (FIG. 12D). A summary of the present example is shown in FIG. 13A.

Half-Life Determination of SABA1.1 and SABA5.1 in Cynomolgus Monkeys

A three week single dose proof of concept study of SABA1.1 and SABA5.1was conducted in cynomolgus monkeys to assess pharmacokinetics at a 1 mgper kg (mpk) dose IV in 2 cynomolgus monkeys. The pharmacokinetics wereevaluated using a quantitative ELISA-based assay that was developed todetect the Adnectin in plasma samples. SABA1.1 has a half-life in therange of 96-137 hours. SABA5.1 has a half-life of approximately 12 hoursand was only measureable in the ELISA up to 120 hours. FIGS. 14 A and Bsummarizes data for these clones and compares data from cynomolgusmonkey.

Example 11 Characterization of SABA1 Binding To Serum Albumin

SABA1.1 and 1.2 Binds to HSA and RhSA

SABA1.2, a “core 1” ¹⁰Fn3 comprising an (ED)₅ extension (SEQ ID NO: 190)bound to human serum albumin (HSA) at neutral pH and 25° C. with anaverage association rate constant (ka) of 8.21E+03 M⁻¹s⁻¹, and anaverage dissociation rate constant (kd) of 4.43E-04 s⁻¹, for acalculated average K_(d) of 55.3 nM (Table 13). For rhesus serum albumin(RhSA), the measured average association rate constant was 6.6E+03M⁻¹s⁻¹, and the dissociation rate constant was 3.78E-03 s⁻¹, giving acalculated average K_(d) of 580 nM. No measurable interaction betweenSABA1.2 and mouse or rat serum albumin could be observed up to 1 μM(Table 13 and FIG. 15). At 37° C., the ka and kd increased between 2 to5-fold, leading to a ˜2-fold increase in affinity for HSA and ½ theaffinity for RhSA (Table 13).

TABLE 13 Kinetic Parameters for SABA1.2 Binding to Albumins, in HBS-PBuffer Temp Albumin (° C.) ka (1/Ms) kd (1/s) KD (nM) Human 25 8.21 ±1.19E+03 4.43 ± 0.65E−04 55.3 ± 13.7 Rhesus 6.60 ± 1.18E+03 3.78 ±0.45E−03  580 ± 62.6 Mouse no observable binding Human 37 3.38E+048.15E−04 24.1 Rhesus 1.89E+04 1.85E−02 977.4 Mouse no observable binding

Additionally, a calorimetric titration was performed to determine thestoichiometry between SABA1 and HSA. For this study, SABA1.1, a “core 1”¹⁰Fn3 comprising a His6 extension (SEQ ID NO: 189), was used. HSA (10 μlper injection of 115 μM protein solution) was injected into thecalorimetric cell containing SABA1.1 at a concentration of 8.1 μM. Theexperiment was performed at 37° C. in PBS buffer pH 7.4. FIG. 16 showsthat SABA1.1 binds to HSA with 1:1 stoichiometry.

SABA1.2 Binds Potently to HSA at Low pH

The long half-life of albumins (e.g., t_(1/2) of HSA is 19 days) is duein large part to the fact that they are recycled from an endocyticpathway by binding to the neonatal Fc recptor, FcRn, under the low pHconditions that exist inside the endosome. As shown in Table 14 SABA1.2potently bound HSA at the endosomal pH of 5.5, suggesting that thet_(1/2) of SABA1, once bound to HSA, would also benefit from the FcRnrecycling mechanism.

TABLE 14 Comparison of Albumin Binding Kinetics at pH 7.4 and 5.5, inMES Buffer albumin pH ka (1/Ms) kd (1/s) KD (nM) Human 7.4 9.26E+033.88E−04 41.9 5.5 9.44E+03 2.70E−04 28.6 Rhesus 7.4 6.16E+03 2.95E−03479 5.5 7.57E+03 2.72E−03 359SABA1.2 Binds to Domains I and II of HSA, but Not Domain III

The binding site SABA1.2 on albumin was mapped to the N-terminal domainsI or II using recombinant HSA fragments and has no detectable binding todomain III (FIG. 17). Because domain III is the domain of HSA thatprimarily interacts with FcRn, it is less likely that SABA1.2 wouldcompete for HSA binding to FcRn, again increasing the possibility offully leveraging the recycling mechanism for enhanced half-life.

Example 12 In Vivo Pharmacology of SABA1.2

A four week single dose pre-toxicology study of SABA1.2 was conducted incynomolgus monkeys to assess pharmacokinetics and immunogenicity at twodifferent dose levels. The pharmacokinetics and immunogenicity were alsoevaluated in a three-week, single-dose pre-toxicology study thatincluded both intravenous and subcutaneous administration arms.Additionally, the pharmacokinetics of SABA1.2 was evaluated in twoseparate, single dose pre-toxicology studies in cynomolgus monkeys usinga quantitative ELISA-based assay that was developed to detect SABA1.2 inplasma samples.

SABA1.2 was administered to monkeys at 1 mpk and 10 mpk IV. As shown inFIG. 18 and the parameters described below, the Cmx and AUC increasedapproximately linear with dose. Non-compartmental analyses usingWINNONLIN® software were performed to evaluate pharmacokineticparameters. The clearance (CL) for SABA1.2 at 10 mpk was 0.15 ml/hr/kg,the beta phase half-life (t_(1/2)) was 143 hours, the volume ofdistribution (Vz) was 30 mL/kg, and total drug exposure (AUCall) was5,609,457 hr*nmol/L (Table 15). The clearance (CL) for SABA1.2 at 1 mpkwas 0.4 ml/hr/kg, the half-life (t_(1/2)) was 124 hours, the volume ofdistribution (Vz) was 72 mL/kg, and total drug exposure (AUCall) was214,636 hr*nmol/L (Table 15).

After SC or IV administration of SABA1.2, the beta-phase pharmacokineticprofiles were similar (FIG. 19). Non-compartmental analyses usingWINNONLIN® software were performed to evaluate pharmacokineticparameters. The clearance (CL) for SABA1.2 at 1 mpk IV was 0.22ml/hr/kg, the beta phase half-life (t_(1/2)) was 125 hours, the volumeof distribution (Vz) was 40 mL/kg, and total drug exposure (AUCall) was357,993 hr*nmol/L (Table 15). The clearance (CL) for SABA1.2 at 1 mpk SCwas 0.32 ml/hr/kg, the beta phase half-life (t_(1/2)) was 134 hours, thevolume of distribution (Vz) was 62 mL/kg, and total drug exposure(AUCall) was 251,339 hr*nmol/L (Table 15). The SC relativebioavailability (F) compared to IV was 0.7.

TABLE 15 Pharmacokinetic Parameters for SABA1.2 in Monkeys Study # 1 2Dose (mg/kg) 1 10 1 1 Route of i.v. i.v. i.v. s.c. administration N 3 31 2 CL (mL/hr/kg) 0.4 0.15 0.22 0.32 Vz (mL/kg) 72 30 40 62 AUCall214,636 5,609,457 357,993 251,339 (hr*nmol/L) beta T_(1/2) (h) 124 143125 134 Bioavailability (F) n/a n/a n/a 0.7

We claim:
 1. A polypeptide comprising a fibronectin type III tenthdomain (¹⁰Fn3) shown in SEQ ID NO:1, wherein the ¹⁰ Fn3 comprises a BCloop selected from SEQ ID NO:2-6, a DE loop selected from SEQ IDNO:7-48, and a FG loop selected from SEQ ID NO:49-59 and wherein thepolypeptide binds the structural epitope of the p19 subunit of IL-23. 2.A polypeptide comprising a fibronectin type III tenth domain ¹⁰Fn3wherein the polypeptide amino acid sequence is selected from SEQ ID NOs:60-100.
 3. The polypeptide of claim 1 or 2 further comprising one ormore pharmacokinetic (PK) moieties selected from the group consisting ofpolyethylene glycol, sialic acid, Fc, Fc fragment, transferrin, serumalbumin, a serum albumin binding protein and a serum immunoglobulinbinding protein.
 4. The polypeptide of claim 3 wherein the PK moiety ispolyethylene glycol.
 5. The polypeptide of claim 1 or 2 furthercomprising a cysteine linker.
 6. The polypeptide of claim 5 wherein thecysteine linker is selected from the group consisting of the amino acidsGSGC shown in SEQ ID NO: 101 and EIDKPCQ shown in SEQ ID NO:
 102. 7. Apharmaceutically acceptable composition comprising the polypeptide ofclaim 1 or 2, wherein the composition is essentially endotoxin free.